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The mechanisms of platelet underproduction in immune thrombocytopenia (ITP) remain unknown. While the number of megakaryocytes is normal or increased in ITP bone marrow, further studies of megakaryocyte integrity are needed. Megakaryocytes are responsible for the production of platelets in the bone marrow, and they are possible targets of immune-mediated injury in ITP. Since the biological process of megakaryocyte apoptosis impacts platelet production, we investigated megakaryocyte DNA fragmentation as a marker of apoptosis from ITP bone marrow biopsies. Archived bone marrow biopsy specimens from ITP patients, bone marrow specimens from controls with normal platelet counts, and bone marrow specimens from thrombocytopenic controls with myelodysplastic syndrome (MDS) were evaluated. Sections were stained with anti-CD61 for megakaryocyte enumeration, and terminal deoxynucleotidyl transferase dUTP nick-end labeling was used as an apoptotic indicator. In ITP patients, megakaryocyte apoptosis was reduced compared to nonthrombocytopenic controls. Megakaryocyte apoptosis was similarly reduced in thrombocytopenic patients with MDS. These results suggest a link between megakaryocyte apoptosis and platelet production.

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by IgG antibodies bound to complexes of platelet factor 4 (PF4) and heparin. The majority of diagnostic tests for HIT rely on an exogenous source of PF4 to identify anti-PF4/heparin antibodies. These include the PF4-dependent enhanced serotonin release assay (PF4-SRA) among others. Using a bacterial expression system, we developed a novel and efficient method of producing recombinant human PF4 (rhPF4) that is biochemically and antigenically similar to platelet-derived human PF4. rhPF4 was produced using the pET expression system in the BL21(DE3) strain of Escherichia coli. The system was optimized for protein expression using isopropyl β-D-1-thiogalactopyranoside at different induction temperatures and incubation times. rhPF4 solubility was improved by using different detergents during cell lysis and by purifying with heparin affinity and ion exchange chromatography. Biochemical characteristics of rhPF4 were investigated using mass spectrometry, SDS-PAGE analysis, and gel filtration chromatography and compared to platelet-derived PF4. Antigenic and functional characteristics of rhPF4 were studied using the anti-PF4/heparin EIA and the PF4-SRA. Using this method, we could produce 11.4 ± 0.6 mg of pure rhPF4 per liter of bacterial culture. Absorbance readings from the anti-PF4/heparin EIA using platelet-derived and rhPF4 were highly correlated (n = 194; r = 0.9545, p 0.0001); and functional release of serotonin in the PF4-SRA induced by anti-PF4/heparin antibodies was similar to either platelet-derived or rhPF4 and heparin (r = 0.9597, p 0.0001). Our method of rhPF4 production is efficient and does not rely on a source of platelets. The rhPF4 purification method described produces greater yields at a lower cost than other current methods. The application of this method can improve the efficiency of biochemical investigations and HIT diagnostic testing by supplying sufficient amounts of PF4.

Human platelets are activated by IgG immune complexes and undergo platelet aggregation and the release of granular contents. Recently, the receptor for IgG on human platelets has been identified and characterised. In contrast, it is not known if rabbit platelets carry Fc receptors. In this report we describe investigations to detect the presence of an Fc receptor on rabbit platelets. The Fc receptor was investigated using functional assays (platelet aggregation and (14)C-serotonin release), a direct binding assay, and an affinity precipitation assay. All of these methods gave consistently positive results using human platelets and consistently negative results using rabbit platelets, indicating that rabbit platelets do not carry Fc receptors. This inconsistency in the interactions of immune complexes with rabbit and human platelets indicates that rabbits are not ideal models for studying a number of human diseases that could be caused by immune complexes, including heparin-induced thrombocytopenia and certain immunological disorders such as systemic lupus erythematosus.

Ancrod, a serine protease purified from the venom of Agkistrodon rhodostoma, has been used as a therapeutic anticoagulant for a number of indications, including replacement of heparin in patients with heparin-induced thrombocytopenia. Ancrod has similar fibrinolytic activity to thrombin, but ancrod specifically cleaves only the alpha chain of fibrinogen, producing the characteristic fibrinopeptides A, AP and AY. Because ancrod has been used in patients with heparin-induced thrombocytopenia, it is important to ensure that ancrod does not directly affect the platelets and potentially increase the hemostatic effect. The effect of ancrod on platelets has not been well established, and there is not agreement in published studies. Additionally, some of the studies are over 15 years old and pre-date sensitive assays such as glycoprotein analysis. For these reasons, we investigated the interaction of ancrod with human platelets using direct and indirect, functional and biochemical techniques. Incubation of platelets with ancrod alone did not induce platelet aggregation or the release of dense-granule contents. Pre-incubation of platelets with ancrod did not augment or inhibit the maximal aggregation achieved with thrombin, nor did it affect the amount of serotonin release from dense granules caused by activation by thrombin. Studies of ancrod-treated platelets using monoclonal antibody-mediated radioimmunoprecipitation demonstrated that high concentrations of ancrod did not cause measurable cleavage of either the glycoproteins Ib-IX or IIb-IIIa. Incubation of radiolabeled platelets with ancrod-treated plasma also had no effect on the platelet glycoproteins, indicating that ancrod does not indirectly affect the major surface receptors. Direct binding studies using radiolabeled ancrod did not demonstrate specific binding to the platelet surface. Together these studies indicate that ancrod does not directly affect nor bind to platelets in vitro. The hypo-coagulant state and subsequent platelet function defect resulting from the use of ancrod appears to be limited to the removal of fibrinogen from the circulation.

A small fraction of patients who receive heparin develop heparin-induced thrombocytopenia (HIT) and, of these patients, a still smaller proportion develop associated thrombotic complications. Heparin-induced thrombocytopenia is caused by the formation of antibodies that bind to specific complexes of platelet factor 4 (PF4) and heparin. However, it remains uncertain why certain patients form these antibodies and develop HIT or why certain patients have thrombotic events. In this report we describe studies on individuals with and without HIT to determined if a potential PF4 polymorphism could explain differences in susceptibility to HIT. In the 10 control individuals and the 10 patients we studied, we did not find a difference in the PF4 sequences. Genetic difference in the PF4 antigenic target does not explain the occurrence of HIT in susceptible patients.