Ivplatelet Factor 4 And The Endothelium

The biochemistry of PF4 and its involvement in HIT is reviewed elsewhere (see Chapter 5). The metabolism of the protein is regulated by its interactions with the endothelium. PF4 is stored in the a-granules of platelets as a tetramer bound to chondroitin sulfate (Barber et al., 1972). The tetramer may dissociate from the GAG as the platelets are activated, but more likely, dissociation occurs subsequent to binding to EC HSPG, which contains a higher charge density. [125I]PF4 is cleared from the circulation with an a-elimination phase approximating 2 min, which primarily represents binding to the endothelium, and a b-elimination phase approximating 40 min, corresponding to uptake and degradation, predominantly by hepatocytes (Rucinski et al., 1986, 1990).

The endothelium binds approximately 50 pmol PF4/105 cells (Rybak et al., 1989). Several classes of binding sites have been identified, including a high-capacity, low-affinity site on HSPG, as well as higher-affinity binding sites involving specific chemokine receptors and certain coagulant proteins (see below). Binding of PF4 to the endothelium is attenuated by pretreatment with heparinase (Marcum et al., 1984), and plasma concentrations are increased 10- to 20-fold after heparin is infused intravenously (Dawes et al., 1982). Binding of PF4 to EC GAGs is electrostatic (Wu et al., 1984) and is independent of the pentasaccharide involved in the binding of AT (Loscalzo et al., 1985). The affinity of PF4 binding to ECs is lower than to purified heparin (Kd = 2-3 mmol/L vs. 2nmol/L, respectively) (Rybak et al., 1989), consistent with the biochemical heterogeneity of vascular matrix. PF4 has a 10- to 100-fold greater affinity for EC HSPG than does AT ( Jordan et al., 1982) and thus markedly attenuates the antiprotease cofactor activity of AT on intact vessels (Busch et al., 1980; Stern et al., 1985).

The involvement of PF4 in hemostasis is mediated in part by charge-dependent interactions with EC proteoglycans (Eslin et al., 2004). Recent in vivo studies utilizing murine PF4 knock-out (mPF4_/~) and human PF4 (hPF4+) transgenic animals show that PF4 helps stabilize clots formed in response to EC injury. Both mice lacking and those that overexpress PF4 show delayed and unstable clot formation, indicating that a narrow range of PF4 concentrations is needed for efficient clot formation (Fig. 1). The defect in thrombus formation in mPF4_/~ can be corrected by infusion of human PF4 or protamine sulfate. On the other hand, the overexpression of PF4 seen with hPF4+ animals as well as infusions of protamine into wild-type animals is associated with impaired thrombus formation, which can be reversed through charge neutralization using heparin (Eslin et al., 2004). These studies suggest that PF4 facilitates clot formation by neutralizing negatively charged surfaces on platelets and ECs, perhaps allowing closer approximation of platelets to each other and to the endothelial lining. In settings where insufficient or excessive PF4 is released, cell surfaces may retain a net negative or positive charge, respectively, which prevents optimal approximation of cellular elements (Eslin et al., 2004).

The existence of an "optimal" concentration range of PF4 for hemostasis mirrors the binding of HIT antibody to complexes formed at various molar ratios of PF4 to heparin (Bock et al., 1980; Greinacher et al., 1994; Rauova et al., 2005). PF4 and heparin form ultra-large complexes (ULCs, MW > 670 kDa) over a narrow range of molar ratios, approximating 1 mole of PF4 tetramer to 1 mole of unfractionated heparin, the ratio at which HIT antibody binding is optimal (Rauova et al., 2005) (see Chapter 5). These ULCs are preferentially recognized by KKO, a murine HIT-like antibody. Recent in vivo studies affirmed the importance of these findings. When KKO is administered to double transgenic mice expressing platelet hFcgRIIA and varying levels of hPF4 (hPF4high/hFcgRIIA, hPF4mid/ hFcgRIIA, or hPF4low/hFcgRIIA), the severity of thrombocytopenia is proportionate

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