Thrombocytopenia

Abbreviations: FcR = Fc receptor; HIT = heparin-induced thrombocytopenia; HITTS = heparin-induced thrombocytopenia with thrombosis; HR = high responder; LR = low responder; PF4 = platelet factor 4

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Heparin was discovered > 80 years ago, (1) and within a short interval it was used as an anticoagulant. (2) Heparin has advantages that led to its widespread use, including its immediate onset of action, its relatively short half-life (3h), and its ability to be reversed using protamine.

In 19580 Weismann and Tobin (3) described paradoxical thrombi during heparin therapy. Ten patients developed severe and sometimes catastrophic arterial occlusion while receiving heparin. Six of the 10 patients died as a result of the thrombosis. (3) The clots were described as being a pale salmon color in appearance and platelet-rich when examined microscopically. An additional 11 patients were described 5 years later by another group. (4) Once again, pale, platelet-rich arterial thrombi were described. And again, no mention of thrombocytopenia was made. (4) We now recognize that although platelet-rich "white clots" can occur, they are uncommon, and the majority of thrombi complicating heparin-induced thrombocytopenia (HIT) are RBC-rich fibrin clots.

In 1973, Rhodes et al (5) described thrombocytopenia as a component of the syndrome. The pathogenic role of heparin was confirmed by the recurrence of thrombocytopenia when a patient was reexposed to heparin. (5) A potential immunologic component was postulated based on the ability of the plasma from these patients to induce platelet aggregation in the presence of heparin.

Our understanding of HIT has evolved dramatically over the past 30 years. In this report, these changing concepts are summarized, with a particular focus on how a better understanding of the pathophysiology of HIT has translated into better treatments.

CHANGING CONCEPTS OF THE CLINICAL EXPRESSION OF HIT

HIT was first described as an arterial thrombotic disorder with the emboli being pale-colored because they were platelet-rich. (2,3) As the recognition of HIT increased across the decades of the 1980s and 1990s, some investigators began to question whether venous thrombi could also be part of the thrombotic syndrome complex. The problem with this hypothesis was that those disorders in which venous thrombi were implicated in HIT, such as in orthopedic surgery, could, by themselves, cause venous thrombi. Boshkov and colleagues (6) addressed this question by relating the type of thrombotic event to the associated medical or surgical situation or surgical procedure. The results of this study suggested that the vascular location of the thrombotic complications (arterial or venous) was related to the underlying vascular damage or associated surgical risk factors. For example, recent arterial surgery or severe atherosclerosis were associated with arterial thromboembolism. Venous thrombi complicating HIT were likely to occur when there were additional risk factors for venous thromboembolism such as recent surgery. (6)

The demonstration that HIT triggered both arterial and venous thrombi was unexpected, since with few exceptions (eg, the antiphospholipid syndrome) thrombi are typically restricted to either the venous or arterial circulations, but not both. Based on these observations, it was postulated that HIT was a panvascular, prothrombotic disorder with localization to the venous or arterial circulation depending on the risk factors present in each circulation. Today, it is acknowledged (7,8) that venous thromboembolic events dominate over arterial thromboembolic events at a ratio of approximately 4:1.

RECOGNITION OF HIT AS AN IMMUNOLOGIC DISORDER

HIT was initially termed heparin-associated thrombocytopenia because it was assumed that the thrombocytopenia was associated, and was not necessarily causally related. Some investigators, including our group, thought that a contaminant within the heparin preparation could be causing the reaction. Indirect support for this hypothesis was provided by the demonstration that different heparin preparations carried different risks for HIT. (9) Heparin is a purified preparation of glycosaminoglycans (ie, long chains of highly charged sugars) that are isolated from beef lung or pork intestine. Unlike most other drugs, heparin is a complex mixture of a large number of related compounds with molecular weights ranging from 1,000 to > 40,000.

Early evidence that HIT was an immunologic disorder included the report of Rhodes et al, (5) which proposed that HIT was caused by a non-complement-fixing, heparin-dependent antibody. Other investigators (10-13) also provided evidence of an immunologic basis for HIT using a variety of assays.

The mechanism by which the heparin and IgG interacted with the platelet soon became a focus for research in many laboratories. Our group showed (14) that standard platelet aggregation was neither sufficiently sensitive nor specific enough to serve as a diagnostic test for HIT. Based on these results, Sheridan et al (15) developed a sensitive and specific test for HIT. By using platelets from healthy donors who had been radiolabeled with a platelet granular component, serotonin, Sheridan et al (15) were able to dramatically increase the sensitivity of the test. The addition of increasing concentrations of heparin to this mixture of the radiolabeled platelets and the test sera from HIT patients resulted in an unusual pattern of platelet activation. A unimodal heparin-dependent pattern of platelet activation was observed, which suggested the possibility of an immune complex disorder. Together, these observations formed the basis of a test for HIT, which is used today.

In 1988, we reported that platelets from different patients with Glanzmann thrombasthenia and Bernard Soulier syndrome, who together lacked glycoproteins Ib, IX, V, IIb, and IIIa, were all capable of being activated by HIT serum and heparin. (16) This indicated that these glycoproteins did not directly participate in the HIT reaction. We observed that purified Fe from nonimmune IgG was capable of blocking the reaction. This observation led us to propose that HIT was caused by heparin-IgG immune complexes that activated platelets through their Fc receptors (FcRs). We were not able to demonstrate that the heparin bound to the patient IgG. This suggested that an unidentified factor was also involved in the formation of the immune complex.

The next focus for research was the identification of the antigenic target of HIT. In 1992, in a significant advance for HIT-related research, Amiral et al (17) demonstrated that it was platelet factor 4 (PF4) that bound HIT IgG. These observations were confirmed by other groups. (18-20) The next focus of research was the clarification of the role of PF4 as the target antigen. It was demonstrated that the optimal target ratio range of PF4 to heparin was 4:1 to 8:1. (19,21,22) The small size of PF4 (70 amino acids) allowed Horsewood et al (21) to synthesize a series of peptides that spanned the entire length of the PF4 molecule. These investigators found that a minimal length of PF4 (specifically, 19 amino acids encompassing the carboxy-terminal peptide including the lysines, which bind heparin) was required for reactivity with the HIT-IgG. But, it was not possible to identify a linear epitope on the PF4 molecule that served as the target antigen. Rather, the results of these studies were consistent with the hypothesis that heparin molecules bundle the PF4, resulting in conformational changes to the molecule, which in turn, become the binding sites for the HIT-IgG. (21) Visentin (23) has reviewed the interaction of heparin with PF4.

CHARACTERIZATION OF THE ANTIBODIES THAT CAUSE HIT

The demonstration that HIT was an immune complex that activated platelets through the FcR confirmed the central role of IgG in the pathophysiology of this disorder. The majority (about 90%) of the HIT-IgG is polyclonal, Ig[G.sub.1], which is expressed alone or with Ig[G.sub.2]. (24) IgM and IgA also have been found in patients with HIT, and some of the enzyme immunoassays measure the levels of these antibodies. (22,25) However, their biological relevance remains uncertain. Amiral et al (26) have also documented a variety of other autoantibodies in patients with HIT, particularly those acting against certain cytokines such as interleukin-8 and neutrophil-activating peptide 2. Additionally, antiphospholipid antibodies are sometimes found in these patients. (27-30) However, the clinical relevance of all of these antibodies remains unknown.

Certain aspects about the antibody (HIT-IgG) that binds to PF4 remain not well understood. It is surprising that this antibody develops so frequently in individuals who are exposed to heparin because heparan sulfate, a glycosaminoglycan that is very similar to heparin, is found naturally in the body. (31-33) Additionally, the explanation for the remarkable rapidity in the production of this IgG antibody, which can form in 5 days, also remains unexplained. Finally, the short persistence of this antibody in patients with HIT also remains unexplained. (34) The progressive decline in the ability to detect the HIT IgG is shown in Figure 1.

[FIGURE 1 OMITTED]

CHARACTERISTICS OF HEPARIN REQUIRED To FORM THE IGG/HEPARIN/P[F.sub.4] IMMUNE COMPLEX

The work of Horsewood et al (21) demonstrated that the HIT-IgG bound to a heparin-induced conformational change in the PF4 molecule. These studies were confirmed and extended by results from as well as Newman and Chong (35) and Suh et al. (36) Suh et al (36) provided data that the target antigens on PF4 were primarily conformationally induced epitopes, but in some patients this could also include compound epitopes made of saccharide plus the PF4. (36)

Newman and Chong (35) provided data on the pathway of activation. They noted that the Fab from the HIT-IgG initially binds to platelet-bound PF4. Subsequently, the Fc region on the IgG molecule binds to the FcR on the same or adjacent platelets, which in turn triggers platelet activation.

The chemical and structural determinants allowing heparin to induce antigenic changes in the PF4 molecule have been studied by a number of investigators. One focus has been to try to identify a heparin species that would neither initiate nor propagate HIT. Our group documented that a variety of other sulfated polysaccharides could substitute for heparin in inducing antigenic changes in PF4. However, there were two key determinants that were required for heparin-induced PF4 antigenicity. First, a certain chain length (ie, approximately > 1,000 d) and, second, a minimal amount of sulfation per saccharide unit were required. (18) These results, which were confirmed by other investigators, (37) suggested that progressively smaller heparin preparations carry a progressively lower risk of HIT. Clinical validation of this potential was provided by the demonstration by Warkentin and coworkers (38) that low-molecular-weight heparin carried a lower risk of HIT than standard unfractionated heparin. Recently, there has been interest in whether the very short heparin-related molecule pentasaccharide will be immunologically irrelevant in initiating or potentiating HIT.

ROLE OF THE PLATELET FcR IN HIT

The platelet FcB (shown schematically in Fig 2) was identified by Karas et al, (39) with further characterization by Rosenfeld et al (40) as well as by our group. (41) This FcR falls within the FcRIIA group of FcR, and carries a low binding affinity and a relatively low copy number per platelet (approximately 1,000 to 2,000 copies per platelet). (39-41) IgG immune complexes, which form in patients with HIT, can occupy the platelet FcR on the same platelet or on crossing platelets. (42) This occupancy is a potent initiator of platelet activation and the platelet release reaction. FcR-mediated platelet activation is independent of the platelet glycoproteins Ib, V, IX, Iib, and IIIa. (16) A study by Polgar and associates (43) has suggested that the adenosine diphosphate receptor also participates in HIT-mediated platelet activation.

[FIGURE 2 OMITTED]

FcRIIA is a linear transmembrane protein carrying two disulphide bonds (Fig 2). There are polymorphic sites at amino acid 131, and a proportion of healthy individuals carry either arginine or histidine at that site. People with the arginine polymorphism (about one quarter of the population) have an enhanced response to murine IgG, but a lower response to human IgG subclass 1. The response to murine IgG has given this polymorphism the designation of high responder (HR). About one quarter of the population is homozygous for histidine at that site and has a lower response to murine IgG (LR), but a higher response to human IgG subclass 1.

This variability of response has led investigators to look for the overexpression of HRs or LRs in patients with or without HIT. Several studies (24,44-48) have produced conflicting results, but for all, the association with HIT was weak. This suggests that the pathologic and biological impact of the LR or HR phenotype in humans who develop HIT is weak.

There are also polymorphisms within the PF4 protein, but these have not been associated with an increased or reduced susceptibility to HIT. (49) The clarification of the pathophysiology of HIT has allowed the development of a precise animal model, which in turn will allow the analysis of novel treatments. (50)

EXPLANATION OF THE ARTERIAL AND VENOUS THROMBOEMBOLIC EVENTS OF HIT

The role for platelets in the pathophysiology of HIT has long been recognized. The HIT-IgG/PF4/ heparin immune complexes are potent platelet activators; however, the explanation for venous thrombotic events as well as arterial thrombotic events in patients with HIT has remained unexplained until recently. It has long been recognized that some patients with severe HIT could have laboratory or clinical evidence of disseminated intravascular coagulation during an acute thrombocytopenic episode. This was noted by Klein and Bell (51) in 1974, and, while uncommon, it is a well-described complication of HIT. I believe that a large percentage of patients with HIT would have disseminated intravascular coagulation, except that the heparin, which is initiating the immnologic reaction, simultaneously is controlling the amount of thrombin that is activated. Evidence supporting the prothrombotic nature of HIT was provided by the study by Warkentin et al (52) of HIT patients in whom heparin therapy had been discontinued and warfarin therapy initiated. These patients developed particularly severe and progressive thrombi. In these patients, the thrombin generated by the HIT became unopposed when (1) the heparin therapy was discontinued and (2) there was an acute, warfarin-mediated decline in the natural anticoagulant protein C. It has also been demonstrated, in vitro, that serum from patients with HIT, in the presence of heparin and platelets, enhances the generation of thrombin from prothrombin following the addition of factor Xa. (53)

An important question facing researchers concerned the mechanism of activation of the coagulation cascade. The following three possible explanations have been put forward: (1) HIT-IgG immune complexes can bind to endothelial cells, activating the coagulation cascade (19,54,55); (2) HIT-IgG can bind monocytes and initiate the release of tissue factor (56-58); and (3) HIT-IgG activates platelets, releasing procoagulant-rich microparticles. (59) As these pathways of coagulation activation in HIT patients were defined, it was also shown (60) that inherited prothrombotic disorders, by themselves, were not an explanation.

It is noteworthy that a variety of drugs are capable of causing drug-induced immune thrombocytopenia. A common and well-studied drug-induced thrombocytopenia is quinine-induced thrombocytopenia. However, with the exception of a few agents that can trigger thrombotic thrombocytopenic purpura, only heparin and related glycosaminoglycans can cause thrombosis as well as thrombocytopenia.

Our group suspected that microparticles could be one explanation for the thrombotic complications of HIT. Blood samples from individuals with acute HIT were studied using flow cytometry. This technique allows one to study very small cellular fragments, which otherwise would not be detected. By using monoclonal antibodies that are capable of specifically recognizing platelet fragments (a monoclonal antibody against glycoprotein IX), we confirmed that, during the acute episode of HIT, there were high concentrations of platelet microparticles that were no longer detectable during convalescence. (59)

The next step focused on replicating these results in vitro. The illustrative experiments are shown in Figure 3. (59) The studies illustrated in Figure 3 document the fact that at maximum platelet activation, as measured by radiolabeled serotonin release from the platelet-dense granules, there is a parallel release of platelet-derived microparticles. High concentrations of heparin did not support this activation step. In other experiments, Warner et al (53) documented that these platelet-derived microparticles were procoagulant, as shown by a significant shortening of the Russell viper venom time. These studies also showed that HIT-induced platelet microparticles can serve as a surface on which activated factor Xa catalyzes the conversion of prothrombin into thrombin. (53) The ability of HIT-IgG to generate microparticles has also been used as the basis for a test for HIT, which measures the generation of platelet-derived microparticles using flow cytometry. (61)

[FIGURE 3 OMITTED]

To better understand these microparticles, Hughes and colleagues (62) examined their morphology using a variety of microscopic techniques. Using confocal microscopy, the release of platelet particles could be observed after the test platelets were incubated with HIT-IgG plus heparin. When compared to a variety of agonists, the reaction was shown to be as intense as that caused by thrombin or calcium ionophore. The use of scanning and transmission electron microscopy showed that these microparticles ranged in size from < 0.1 to 1.0 [micro]m in diameter. Based on serial studies, we were able to propose that the HIT-mediated platelet activation resulted in the formation of localized points of swelling on the platelet body, with the formation of well-defined buds. These platelet buds are released from the platelets themselves to form the microparticles, which characterize the HIT (62) (Figs 4-6). Together, one can conceptualize the pathophysiolog) of HIT, as shown in Figure 7.

[FIGURES 4-7 OMITTED]

TREATMENT STRATEGIES FOR HIT BASED UPON THE RESULTS OF LABORATORY AND CLINICAL OBSERVATIONS

The treatment of HIT has included the use of numerous medications, many of which are no longer used. Ancrod is one such agent. (63) This snake venom extract is capable of cleaving fibrinogen and causes rapid anticoagulation by lowering the concentration of fibrinogen in the blood, which prevents the ability of a clot to continue to incorporate fibrin into the thrombus. While ancrod proved useful in some patients with HIT, it was not universally successful. (63) More importantly, the recognition of the pivotal role of thrombin generation in the pathogenesis of HIT suggested that the central pillar of treatment for patients with HIT should be a direct thrombin inhibitor.

For many years, it was suggested that an important first step after diagnosing HIT was the discontinuation of heparin therapy as soon as possible. Ironically, for some patients, the continuation of heparin therapy does not result in clinical worsening, and the thrombocytopenia can rarely resolve. In other patients, the thrombotic events would worsen when the heparin was discontinued. Nonetheless, it is generally accepted that therapy with heparin, both unfractionated and low-molecular-weight, be discontinued as soon as possible.

Until recently, the commonly used approach was to discontinue therapy with heparin and immediately institute use of an oral anticoagulant, warfarin. However, dramatic clinical observations by Warkentin and coworkers (52) challenged this approach. They described patients with HIT who had a stable venous thromboembolism, often of the lower limb. These patients had a catastrophic outcome when therapy with heparin was discontinued and that with warfarin was initiated. (52) All had a characteristic laboratory and clinical picture, which suggested that they had a novel syndrome, which was termed warfarin-induced venous gangrene. (52)

The clinical presentation included the following. First, the patients had relatively stable HIT and an underlying venous thrombus. When heparin therapy was discontinued and warfarin therapy was introduced, there was an acute and dramatic onset of venous gangrene within days. Some of the patients had peculiar "scalded skin" lesions on the affected limbs with blistering resembling a severe burn injury. Inspection of biopsy specimens demonstrated the presence of microthrombi of the arterial and venous capillaries. (52)

The extensive nature of the thrombi throughout the microcirculation and macrocirculation explains why many lost their limbs despite aggressive management. The patients also had a characteristic laboratory presentation. Virtually all patients had a disproportionately high international normalized ratio (postulated to be related secondarily to the decline in vitamin K-dependent factor VII) and an exceptionally low level of the natural anticoagulant protein C.

It was postulated (52) that the very low levels of protein C and factor VII were due to underproduction (both proteins have a short half-life of 6 to 8 h) plus the consumption of these vitamin K-dependent factors. These observations have led to recommendation that warfarin not be used during acute episodes of HIT. Warfarin should be administered cautiously only after the patient with HIT has begun to recover completely, as manifested by a return of the platelet count to normal levels, and when the patient has been adequately anticoagulated with an alternative parenteral agent.

In some countries, but no longer in the United States, danaparoid (Organon; Roseland, NJ), a heparin-related substance, has been used. This glycosaminoglycan is primarily made from heparan sulfate, dermatan sulfate, and chondroitin sulfate. (64) It is estimated that there is approximately 20 to 30% cross-reactivity of this heparinoid with HIT-IgG, but in most patients (65,66) there are not major adverse effects. However, some patients will still have a reaction to the anticoagulant. (67)

A better understanding of the pathophysiology of HIT, and specifically the role of excess thrombin generation during the acute episode, led to the evaluation of direct thrombin inhibitors as treatment for HIT with or without associated thrombosis. Two distinctly different thrombin inhibitors, lepirudin (a recombinant version of the protein hirudin) and argatroban (a small, synthetic molecule), have been evaluated in several prospective studies. (68-73) The strengths and weaknesses of these trials have been recently reviewed. (74) All studies had the major advantage of being prospective in nature. The lepirudin studies (70-72) had the diagnosis confirmed serologically in treated patients. The argatroban studies (68-69) relied on a clinical diagnosis of HIT. (Subsequent serologic test results demonstrated a 50 to 65% positive serology rate in the studies.) All studies had a remarkable consistency. The direct thrombin inhibitors argatroban or lepirudin, relative to historical control therapy, produced a highly significant reduction in new thromboembolic events in patients with HIT. However, there was no overall reduction in death, observations suggesting that, for many patients with HIT, the reaction is so severe that by the time treatment is initiated irreversible events have occurred. For example, the prospective studies documented that, even with prompt treatment with a direct thrombin inhibitor, 10 to 20% of patients with HIT will still die, and for those who survive about 5 to 15% will require an amputation or will have had a severe thromboembolic event. (74) Consequently, future research should focus on identifying particular subsets of patients who have exceptionally severe disease as well as considering additional treatments for those who are at the highest risk.

The analysis of large groups of patients with HIT has revealed several aspects of the syndrome complex that impacts on therapy. For example, although the prothrombotic nature of HIT has long been recognized, it has only recently been recognized that patients without all underlying thrombosis are at particularly high risk of progressing to a thrombosis if heparin therapy is discontinued and no other alternate therapy is instituted. In a recent analysis of retrospective and prospective studies, (74) it was shown that 20 to 50% of patients with HIT and no apparent thrombosis will have a thrombotic complication within the next month, most frequently within 1 to 2 weeks after discontinuing the heparin therapy. This has led to the recommendation (74) that an alternate therapy with a rapidly acting anticoagulant should be instituted as soon as possible in a patient with HIT, even if that patient has no evidence of thrombosis.

The same analysis (74) reviewed the use of lepirudin and argatroban in patients with HIT (ie, HIT and thrombocytopenia only) as well as in those with HIT with thrombosis (HITTS) [ie, HIT plus an associated thrombotic event]. Although no report has described the use of lepirudin in HIT patients, it is likely that the antithrombotic effects are similar to that of argatroban in HIT patients, which in turn are similar to both agents in HITTS patients. Specifically, the studies do not document a reduction in death, although individually and together they show a reduction ill new thromboembolic events when the thrombin-specific inhibitor is used for treatment (Fig 8).

[FIGURE 8 OMITTED]

The analysis did document one difference between lepirudin and argatroban. Lepirudin was associated with a higher risk of bleeding ill the studies evaluating its use compared with argatroban. It is possible that this is a true difference, or it may reflect differences in study design (Fig 9).

[FIGURE 9 OMITTED]

Lepirudin also has been associated with the formation of antibodies. These occur frequently (ie, in as many as 50% of patients receiving lepirudin for long intervals) and typically do not cause significant problems except some alteration in drug pharmacokinetics. (75) Rarely have fatal allergic reactions been described. (76) Antibodies to argatroban have not been described. (77)

CURRENT RECOMMENDATIONS FOR MANAGEMENT OF PATIENTS WITH HIT

The diagnosis of HIT should be made clinically and requires a high index of suspicion. Treatment should be instituted and serologic testing performed. As described elsewhere in this Supplement, there is not a diagnostic laboratory test of choice, and each laboratory approach offers distinct advantages and disadvantages. Both the enzyme immunoassays and the functional assays have a very high sensitivity for HIT. (78) For most physicians, the diagnosis is made based on a high index of suspicion and by using clinical criteria, with a negative test result, particularly if the test has been repeated, making the diagnosis distinctly unlikely.

HIT is a common disorder, and its frequency is related to the type of heparin received, the dosage of heparin received, the animal origin of the heparin, and the clinical situation. Unless there has been a recent (ie, < 3 to 4 months) exposure to heparin, the onset of HIT occurs after 5 days and can be insidious or precipitous, with the occurrence of isolated thrombocytopenia or thrombocytopenia plus concomitant thromboembolic events. (34) Typically, venous thrombi predominate at a rate of 4:1, but the general observation is that the more unusual the thrombus, the more that HIT should be considered.

Treatment with all types of heparin should be discontinued as quickly as possible, and a direct thrombin inhibitor should be used. The choice depends in part on the familiarity of the physician with the agent and the clinical situation. Because argatroban is metabolized by the liver, it can be used safely in patients with renal failure, but should be used with caution in patients with hepatic dysfunction. The reverse is true for lepirudin. The direct thrombin inhibitor should be administered for a minimum of 7 days or until the platelet count has risen to normal, and then therapy with warfarin should be introduced very slowly and at a low dose. Until further studies are completed, we suggest a longer (at least 5 days) rather than a shorter overlap period.

Both prospective and retrospective studies (7,8) have shown that there is a high risk of isolated thrombocytopenia caused by HIT progressing to thrombotic complications, and currently we would manage patients with this condition with a direct thrombin inhibitor. Because the risk of thrombosis can persist for weeks, many physicians would initiate warfarin therapy in these patients for several months. Although some reports (34,79) have noted that heparin can be tolerated for a brief period in patients in whom levels of circulating HIT-IgG are no longer detectable, the reexposure of patients with a history of HIT to heparin is not generally advised. If possible, these patients should be managed with a direct thrombin inhibitor. This general recommendation may evolve as more is learned about the transient nature of HIT antibodies and the role of immune memory in HIT.

* From McMaster University, Hamilton, ON, Canada. Some of the studies described in this report were supported by a grant from the Heart and Stroke Foundation of Ontario. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org).

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Correspondence to: John G. Kelton, MD, McMaster University, 1200 Main St West, Room 2E1, Hamilton, ON, L8N 3Z5, Canada; e-mail: keltonj@mcmaster.ca

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