PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium

Abstract

We previously reported that only a subpopulation of PAR-1-stimulated platelets binds coagulation factor IXa, since confirmed by other laboratories. Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by measuring fluorescence changes in Fura Red/AM-loaded platelets following PAR-1 stimulation. While fluorescence changes in all platelets indicated calcium release from internal stores and influx of external calcium, a subpopulation of platelets displayed a pronounced increase in calcium transients by 15 seconds and positive factor IXa binding by 2 minutes, with calcium transients sustained for 45 minutes. Pretreatment of platelets with Xestospongin C to inhibit IP3-mediated dense tubule calcium release, and the presence of impermeable calcium channel blockers nifedipine, SKF96365 or LaCl 3, inhibited PAR-1-induced development of a subpopulation with pronounced calcium transients, factor IXa binding, and platelet support of FXa generation, suggesting the importance of both release of calcium from internal stores and influx of extracellular calcium. When platelets were stimulated in EDTA for 5 to 20 minutes before addition of calcium, factor IXa binding sites developed on a smaller subpopulation but with unchanged rate indicating sustained opening of calcium channels and continued availability of signaling elements required for binding site exposure. While pretreatment of platelets with 100 μM BAPTA/AM (K d 160 nM) had minimal effects, 100 μM 5, 5′-dimethylBAPTA/AM (K d 40 nM) completely inhibited the appearance and function of the platelet subpopulation, indicating the importance of minor increases of cytoplasmic calcium. We conclude that PAR-1-stimulated development of factor IXa binding sites in a subpopulation of platelets is dependent upon release of calcium from internal stores leading to sustained and pronounced calcium transients.An essential event in the hemostatic response to vascular injury is the assembly of the factor X (FX) 1 activating complex on the surface of activated platelets (1,2). The assembly of this important enzymatic complex requires the exposure of coagulation protein binding sites on the surface of platelets activated with thrombin or collagen but not with adenosine diphosphate (3-6). All proteins required for physiologically relevant platelet-supported FX-activation, including the enzyme factor IXa (FIXa), the cofactor factor VIIIa (FVIIIa) and the substrate factor X (FX), must be bound to their respective receptors on activated platelets (7). Thus, the zymogen FIX binds with high affinity (K d ~2.5 nM) to a discreet number of platelet receptors † This study was supported by research grants from the National Institute of Health: HL70683, HL46213 and HL74124. *To whom correspondence should be addressed: Fredda London, Ph.D., Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, Tel: 215-707-4458; Fax: 215-707-3005;...

Two Types of Procoagulant Platelets Are Formed Upon Physiological Activation and Are Controlled by Integrin α IIb β 3

“…This phenomenon is universal for all strong platelet agonists. Importantly, in contrast to previous experience, 10,11 this new pathway seems not to require sustained high calcium levels: even after 40 minutes, when calcium in all platelets has reverted to the basal state, we still see formation of the PS+/Ca-platelets.…”

“…In contrast to previous reports, 2,10,11,17 not all of the PS-positive platelets sustained a high level of intracellular calcium after an initial increase. After several minutes from the beginning of activation, we could observe formation of 3 different subpopulations: (1) PS-negative platelets with low intracellular calcium level (PS-/Ca-), (2) PS-positive platelets with sustained high calcium level (PS+/Ca+) similar to those described previously, 2,10,11,17 and (3) a new subpopulation of platelets that had high PS together with unexpectedly low calcium (PS+/Ca-).…”

“…6 These platelets are highly effective in binding coagulation factors and accelerating membrane-dependent coagulation reactions, 8,9,11 but their integrin α IIb β 3 is inactivated, 12,17 which reduces their chances of being accumulated into thrombi or hemostatic plugs. Their significance for hemostatic and thrombotic events is not well understood: their deficient production caused by inhibition of the cyclophilin D-dependent necrotic pathways is not associated with bleeding but rather with increased thrombosis.…”

Modulation and pre-amplification of PAR1 signaling by ADP acting via the P2Y12 receptor during platelet subpopulation formation

“…One of them has high phosphatidylserine (PS) expression on their surfaces, thereby it is highly procoagulant [3,4]. Procoagulant platelets have high level of intracellular calcium [5], which is mobilized by activation of the 1,4,5-trisphosphate (IP 3 ) signaling pathway. IP 3 formation induced by thrombin is in turn mediated by phospholipase Cβ which can be activated by Gαq proteins associated, in particular, with protease-activated receptors [6] (PAR 1 and PAR 4 ).…”

Important Regulatory Role of Activated Platelet-Derived Procoagulant Activity in the Propagation of Thrombi Formed Under Arterial Blood Flow Conditions

“…Indeed, a previous report demonstrated that factor IXa binding to platelet and generation of activated factor Xa was prevented by the putative Ca 2+ channel-blocker of lanthanum chloride. 47 Unlike in vivo animal experiments 33,48 or atherothrombosis in humans, 2,3,6 our experiments did not include tissue factor originating from the vascular wall or ruptured atheroma. Thus, tissue factor, if it played a role in the generation of fibrin in our experiments, is limited to a blood-borne one.…”