Platelet Factor 4 in Cardiovascular Disease

This issue of Seminars in Thrombosis and Hemostasis reviews the role of platelet factor 4 (PF4) in cardiovascular diseases. PF4 is a protein of the chemokine family that is stored in megakaryocytes and platelets and released upon platelet activation. Probably its most important role is in the development of heparin-induced thrombocytopenia (HIT), when PF4 forms complexes with heparins, especially unfractionated heparin. Antibodies that form to these complexes not only bring about the drop in platelet counts, but also lead via multiple mechanisms to serious thromboembolic complications. The issue also reviews the management options that are presently available to keep patients with HIT adequately anticoagulated during cardiac surgeries and during percutaneous cardiac interventions. Articles provide important diagnostic considerations of HIT and review mechanisms by which these antibodies provoke their devastating effects. The potential use of PF4 or PF4 fragments to neutralize the anticoagulant effects of heparin is indicated, as is its potential application as an inhibitor of angiogenesis.

In the first article, Warkentin provides an overview of the HIT syndrome, caused by heparin-dependent platelet-activating IgG antibodies that create a state of hypercoagulability. In addition to activating platelets, these antibodies also activate endothelial cells and monocytes that further contribute to increased thrombin generation. Patients may present with thrombocytopenia only, but others may develop thromboembolic complications, arterial and venous. Additional factors, such as vascular diseases, intravascular catheters, and others, further foster thromboembolic events. Prompt identification and proper management can reduce morbidity and mortality in these patients. The immediate discontinuation of all heparin-like material is imperative and other thrombin inhibitors must be employed. Oral anticoagulants should be avoided because the early drop in protein C can lead to coumarin-induced limb gangrene. Danaparoid, a heparinoid, is one alternative anticoagulant; lepirudin and argatroban are other modalities. This comprehensive review expertly lays the foundation for the subsequent contributions.

In the next article, Smith and coworkers present three cases of HIT in cardiac patients. These cases illustrate the severity and complexity of this syndrome. The first demonstrates the importance of serial platelet counts when patients are treated with unfractionated heparin (UFH) and stresses the need for vigorous clinical monitoring of these patients. The initial diagnosis of HIT is still based on clinical impressions and drops in platelet counts for no other reasons than exposure to UFH. The second case reiterates the importance of clinical impressions because this patient had a negative test result with the commonly used platelet aggregation procedure. In addition, this patient was receiving UFH and also other medications that can be associated with thrombocytopenia. The third case illustrates that HIT can be encountered in patients receiving low-molecular-weight heparins (LMWHs). It demonstrates that, although the incidence of HIT with the use of LMWHs is considerably lower, compared with UFH, HIT can develop with this form of treatment. Patients should be taken off all heparin-like medications.

In the third article, Mattioli examines the frequency of HIT in patients with cardiovascular diseases. After a brief discussion of the pathophysiology and the clinical presentation of HIT, the author describes the frequency with which the syndrome is encountered in patients receiving LMWHs. While the incidence is clearly lower than with UFH, there is no difference in the clinical presentation or in the degree of thrombocytopenia. A large number of patients undergoing cardiopulmonary bypass surgery develop HIT antibodies (27 to 50%), but only few proceed to have thrombocytopenia. Many patients, due to previous exposure to heparins, already have antibodies before surgery, and again most do not present with HIT. The reasons for this observation are not entirely understood. The author also addresses the issue of anticoagulation of patients with a history of HIT.

Call and colleagues review the rational for using direct thrombin inhibitors in the management of HIT and summarize the published experiences with lepirudin and argatroban. Lepirudin is a recombinant version of hirudin, the anticoagulant found in the saliva of leeches. Lepirudin neutralizes thrombin that is circulating freely and that is bound to fibrin in clots. Two clinical trials and one meta-analysis revealed superior effects when compared with historical controls. There was a higher incidence of bleeding in the lepirudin group. At this time there is no antidote available, but the half-life of lepirudin is short. The second drug reviewed is argatroban, a synthetic compound that binds directly to the active site of thrombin. Again, both freely circulating and clot-bound thrombin are neutralized. Two large clinical studies have been performed with argatroban and both demonstrated superiority when compared with historical controls. The authors provide detailed information on the use of these two compounds in HIT patients, discuss ways to monitor them to assess safety, and provide suggestions on how to manage patients in whom the drug effects have to be reversed.

In the next article Nikolsky and Dangas discuss the present status of managing patients with HIT who require percutaneous coronary interventions (PCI). The hypercoagulable state produced by HIT together with the mechanical plaque disruption during the PCI place these patients at high risk for thrombosis. Since heparins cannot be used, direct thrombin inhibitors are the drugs of choice. Several compounds have been developed and lepirudin and argatroban have been most widely tested. Two small trials have shown efficacy of lepirudin in preventing coronary reocclusion. One multicenter trial using argatroban as an anticoagulant has also revealed efficacy. Less information exists on bivalirudin, a small peptide that blocks the active site of thrombin. The defibrinogenating compound ancrod has also been used. Data on mechanisms of action, side effects, and other safety issues are reviewed.

Greinacher expertly reviews the use of direct thrombin inhibitors in HIT patients who require cardiovascular surgery. It is well recognized that cardiopulmonary bypass (CPB) surgery using UFH is associated with a high degree of heparin-dependent antibody formation and 25 to 50% of all patients test positive postoperatively. When UFH is continued postoperatively, about 1 to 3% of all CPB patients develop HIT with potential limb loss or death. Even LMWHs induce HIT in these patients, although this is less likely than with the use of UFH. The diagnostic signs of HIT, the incidence of thromboembolic events, the need for laboratory monitoring, and the interpretation of laboratory results are discussed. Alternative anticoagulants are reviewed for managing these patients; direct thrombin inhibitors, such as lepirudin and argatroban, appear to be the drugs of choice, although danaparoid has also been used. Dosing, methods of monitoring, side effects, and proper use of these compounds are extensively described.

In article seven, Maroo and Lincoff provide an overview of the REPLACE-2 trial. This multicenter, randomized, double-blind, and active controlled study compared the use of UFH plus a glycoprotein (GP) IIb/IIIa inhibitor with bivalirudin plus optional GP IIb/IIIa blockade in several thousand patients requiring PCI. The data were evaluated for death, myocardial infarction, and urgent repeat revascularization after 30 days and after 6 months. In-hospital major bleeding was also evaluated after 30 days. The patients treated with bivalirudin had a superior outcome in the first three parameters at both time points and had a significant reduction in bleeding events. The authors extensively review the protocol and the results of the study and also outline the limitations of the investigation.

Next, Merry presents the use of bivalirudin and other direct thrombin inhibitors as anticoagulants in coronary bypass surgery. Alternatives to UFH are needed for patients who have allergies to protamine. The choice of the drug may not only determine complete anticoagulation during the bypass procedure but may also impact graft patency. After reviewing the challenges presented by coronary surgeries and the wide use of UFH as anticoagulant, the author reviews other anticoagulant options, such as LMWHs, heparinoids, defibrinogenating agents, antiplatelet drugs, and direct thrombin inhibitors. The status of each of these agents is presented. Merry also summarizes the results of a completed pilot study in which bivalirudin was used in off-pump coronary artery bypass surgeries. Compared with UFH, bivalirudin did not increase the bleeding tendency in these patients and revealed, after 3 months’ follow-up, improved graft flow.

In the next article, Mascelli and coworkers report on a study performed on patients who were part of the GUSTO IV-ACS trial. The objective was to identify the frequency of the presence of PF4/heparin antibodies in patients previously exposed to UFH during myocardial infarction (MI), coronary bypass surgery, or PCI. An immunological assay with a specificity of 99% was employed. Twenty-three of 218 patients (10.6%) were found to have the antibodies. These patients were more likely to suffer death or acute MI than their negative cohort. These patients also had significantly higher levels of adhesion molecules, such as sCAM-1 and sICAM-1. The presence of these antibodies was a strong predictor of 30-day MI. The data indicate that prior exposure to UFH leads to PF4/heparin antibody formation that impacts the development of acute MIs.

Sachais and colleagues report on the interaction of PF4 with components of the vessel wall. PF4 has a high affinity for negatively charged molecules, especially heparan sulfate. The authors propose that the importance of PF4 and the basis for its presence in α-granules of all mammalian platelets is the neutralization of heparan sulfate on the surface of endothelial cells, thereby optimizing local thrombus formation. This binding may also be the basis for the angiostatic and pro-atherogenic activities of PF4. Binding of PF4 to other negatively charged molecules, such as oxidated low-density lipoproteins, activated protein C/thrombomodulin complexes, fibroblast growth factors, and vascular endothelial growth factors may further contribute to its wide spectrum of biological and pathobiological activities.

In the next contribution, Francis comprehensively and critically reviews the laboratory tests used to confirm the clinical diagnosis of HIT. While there is as yet no assay that identifies the antibodies with 100% accuracy, a number of functional and immunological test systems are marketed. The functional assays rely on platelet activation and have greater specificity. However, they require normal donor platelets that can be difficult to obtain, and the tests are relatively unstandardized. The immunological assays which detect the presence of antibodies, regardless of their functional ability, are better standardized and do not require normal donor platelets. These tests, though, may give positive results without clinical evidence of HIT. This is especially the case in patients following CPB surgeries. In the absence of more rapid test strategies, the diagnosis of HIT is still primarily based on clinical evidence. Laboratory testing should be used to confirm HIT in the proper clinical setting. This article presents comprehensive and practical information on the diagnosis of HIT.

Mixon and Dehmer review studies in which PF4 was used to neutralize the anticoagulant effects of heparin. The heparin-neutralizing capacity of PF4 has long been known, but protamine sulfate was clearly the most widely used compound. Protamine administration can be associated with a number of serious side effects and the authors list those in detail. PF4 is an attractive alternative because it is associated with only few complications and initial animal and human experiments yielded encouraging results. These data led to the development of recombinant PF4 (rPF4). rPF4 underwent extensive animal studies and a few clinical investigations. The data demonstrated that rPF4 is indeed superior to protamine in neutralizing the anticoagulant effects of heparin. Unfortunately this development was not further pursued because other alternatives became available, most notably heparinases. These will likely replace protamine in the future.

In the last article, Bikfalvi describes the role of PF4 in angiogenesis. PF4 inhibits endothelial cell proliferation and migration and angiogenesis. Several fragments of PF4 and several derived molecules have been prepared that also show angiogenesis-inhibiting effects. PF4’s action appears to be manifold, but blockade of growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) seems to be rather prominent. At least three mechanisms are discussed: competing of PF4 with the cooperative effects of heparan sulfate proteoglycans and growth factors, direct interaction of PF4 with angiogenesis factors, and activation of specific cell surface receptors by PF4. It is speculated that PF4 regulates collateral vessel formation, atherosclerotic plaque neovascularization and, perhaps, endothelialization of stents. The hope is expressed that PF4 or possibly some of its derivatives might be developed as therapeutic agents for the regulation of angiogenesis.

I offer many thanks and appreciation to all authors for their informative contributions to the subject matter. Special thanks go to Dr. David Sane, the guest editor of these articles, for assembling such a valuable and informative issue.