Arthur P. Bode

Expression of coagulant activity in human platelets: release of membranous vesicles providing platelet factor 1 and platelet factor 3.

The relationship between the appearance of membrane-associated factor V-like activity (platelet factor 1, PF1) and phospholipid-like catalytic activity (platelet factor 3, PF3) has been examined, in vitro, in collagen-stimulated, human platelets. Both activities increased 7 fold upon collagen treatment relative to stirred controls. After sedimentation of stimulated platelets, 31% of total PF1 and 41% of PF3 remained in the supernatant fraction. PF1 eluted from a Sepharose CL-4B column in the same void volume fractions as PF3, phospholipid, and vesicular particles. These fractions had roughly 100 fold (lipid basis) or 1000 fold (protein basis) enhanced specific activity when compared to the stimulated platelet suspension. Freeze-fracture electron microscopy demonstrated that these void volume fractions contain two populations of membranous vesicles (80-200 nm and 400-600 nm in diameter). Upon centrifugation of the void volume fractions, PF1 and PF3 activities, phosphate-containing material, and ultraviolet-absorbing material all sedimented at the same rate, indicating that PF1 and PF3 are activities associated with one or both of the platelet-derived vesicle populations. Finally, we examined the effects of inhibitors on the appearance of PF1, PF3, platelet factor 4, total intrinsic factor V activity, and serotonin as well as on platelet aggregation. These studies suggest that the collagen-stimulated release of PF1 and PF3 is not coupled to either platelet aggregation or PF4 release but is probably a separate phase of the release reaction.

Association of factor V activity with membranous vesicles released from human platelets: Requirement for platelet stimulation

The membrane-associated factor V-like activity (platelet factor 1, PF1) and the phospholipid-like catalytic surface activity (platelet factor 3, PF3) were studied in human platelets from normal and two factor V-deficient donors. Collagen stimulation or mechanical disruption of gel-filtered platelets was necessary for the expression of significant amounts of PF1 and PF3. Stimulation was also necessary for the uptake of factor V or Va by PF1-deficient platelets from the factor V-deficient donors. The activity of PF1 was also generated by association of factor V or Va with membrane-rich fractions obtained by gel filtration of the supernatant from collagen-stimulated or frozen-thawed PF1-deficient platelets. The amount of PF1 obtained by such all-or-none binding experiments was directly proportional to the amount of PF3 already expressed in the platelet preparation. These data have been summarized in terms of a hypothesis which views PF1 and PF3 to be activities associated with membranous vesicles released from platelets only after stimulation.

Analysis of Platelet Factor 3 in Platelet Concentrates Stored for Transfusion

Abstract. The amount of platelet factor 3 (PF3) activity expressed in stored platelet concentrates (PC) was measured in conjunction with extracellular LDH levels. Standard manual techniques for preparation of PC resulted in PF3 and LDH levels remarkably higher than those observed in PC prepared by apheresis or special manual plateletpheresis. During storage of PC, PF3 activity rose 2‐ to 10‐fold, while LDH levels rose <2‐fold over starting values. Loss of LDH and appearance of PF3 expressed as a percent of total per platelet were significantly correlated only in standard, manual PC. Approximately half of the PF3 activity observed in any type of PC remained in the supernatant plasma after centrifugation. Upon gel filtration, the supernatant PF3 activity eluted in a high molecular weight peak containing phosphate and light‐scattering material. Our findings indicate that platelets in standard, citrated PC express PF3 in amounts that approach that of frozen‐thawed (lysed) platelets; however, the manner in which the PF3 activity appears suggests that stored platelets undergo a combination of activation and damage processes.

Assessment of platelet activation by coronary sinus blood sampling during balloon angioplasty and directional coronary atherectomy

Three markers of platelet activation (platelet-derived microparticles, fibrinogen binding and expression of P-selectin) were assessed by flow cytometry during diagnostic coronary angiography and therapeutic coronary interventions. In 24 patients undergoing diagnostic angiography, blood was collected to determine if our sampling techniques or coronary angiography caused platelet activation. Changes during diagnostic angiography were used to establish baseline values and interpret changes during coronary interventions. In 21 patients, blood samples were obtained at 5 time points during percutaneous transluminal coronary angioplasty (PTCA) (n = 17) or directional coronary atherectomy (DCA) (n = 4). During coronary interventions, mean values for the percentage of platelets expressing P-selectin or binding fibrinogen increased, but with considerable variation among patients. Individual responses for platelet activation markers in each patient were characterized using a twofold increase to indicate elevation related to the intervention. Patients were classified as having complicated or uncomplicated procedures based on the presence of acute closure, dissection, or thrombus observed by angiography. There were no differences in the percentage of elevated markers between patients with uncomplicated (12.5%) and complicated (19%) PTCA procedures. However, patients treated with DCA had more elevated markers (38%) than those treated with PTCA (15%) (p = 0.04). Our data suggest that the extent of platelet activation in individual patients cannot be predicted by common angiographic findings or complications. More markers of platelet activation were present after DCA and may reflect a greater degree of vascular trauma associated with this procedure.

Generation and Degradation of Fibrinopeptide A in Stored Platelet Concentrate

Abstract. The levels of fibrinopeptide A (FPA) were measured in samples from stored platelet concentrates (PC) by radioimmunoassay. In 27 standard, citrated PC, the mean FPA was 13.6 ng/ml, which is elevated 5–6 ′ over background levels. This value did not change significantly over a 7‐day storage period. Addition of PGE‐1 and theophylline resulted in higher initial levels of FPA (18.0 ng/ml) and a pronounced rise during the storage period (to 43.4 ng/ml by day 10). In contrast, addition of a thrombin inhibitor, D‐phenylalanyl‐L‐prolyl‐L‐arginine chloromethyl ketone – 2 HCl (PPACK) or hirudin, resulted in lower initial levels of FPA relative to standard, citrated PC and a slow increase over time. Introduction of exogenous FPA into citrated PC resulted in a predicted elevation of FPA levels followed by a rapid loss of immunoreactivity (t1/2 = 18 h). Addition of PPACK did not affect this fall‐off. However, PC prepared and stored in the presence of PGE‐1 and theophylline showed a much slower fall‐off of exogenous FPA (t1/2 = 38 h). These data indicate that FPA levels in samples from citrated PC represent a dynamic balance between generation and degradation processes and, thus, the data above underestimate the amount of thrombin activity present in stored, citrated PC.

Differentiation of factor V-like coagulant activity from catalytic phospholipid-like surface activity in membrane fractions derived from human platelets

Abstract To investigate the role of the platelet membrane as a catalytic surface in thrombin generation, a relatively physiologic clotting assay was devised that was based on the kaolin-activated partial thromboplastin time. Frozen-and-thawed lysed platelets from normal and Factor V-deficient donors were used as a model for maximally activated normal or V-deficient platelets. An aqueous dispersion of brain phospholipids was employed as an analogue of the lipid portion of a platelet membrane. This test system made it possible to distinguish quantitatively between the contributions of an intrinsic platelet-associated Factor V-like activity (platelet factor 1 or PF1) and phospholipid-like catalytic surface activity (platelet factor 3 or PF3). The degree to which a particular membrane preparation shortened the clotting time was related to its overall catalytic surface activity which was the sum of the contributions of PF1 and PF3. In platelet preparations, both PF1 and PF3 were associated with a membrane-rich fraction that was not easily sedimentable at 12,000g-min. The log-log slope obtained for dilutions of a membrane preparation was taken as catalytic surface effectiveness and was dependent upon the parametric level of Factor V in the test system. The observed dependence was different for each membrane preparation. Effectiveness appeared to be a combined function of both the availability and occupancy of Factor V binding sites as well as the specific affinity of such sites for Factor V(Va).

The platelet membrane as a catalytic surface in thrombin generation : availability of platelet factor 1 and platelet factor 3

The contribution of platelets to blood coagulation can be described in general terms as: the possible site of activation of certain clotting factors; a surface upon which certain clotting factors may selectively bind; and a potential source of coagulant activities.l Presumably, the circulating platelet must be activated before it will participate in coagulation. In response to any of several agents (thrombin, ADP, collagen, epinephrine, etc.) platelets undergo morphological and biochemical changes that coincide with the release of several intracellular constituents.2-4 The appearance (or availability) of platelet coagulant activities seems to follow this release reaction, although some evidence suggests that the appearance of platelet coagulant activities may be associated with the small amount of platelet lysis that accompanies the release r e a ~ t i o n . ~ The contribution of the platelet membrane as a catalytic surface in enhancing thrombin generation can be measured as a clot-promoting activity, which has been defined operationally as platelet factor 3 or PF3. An operational definition has been necessary because PF3 does not appear to result from the contribution of a single molecular component,G but is believed to be a generalized chemical property of negatively charged lipids (for reviews see References 7-9). Whether PF3 is an acquired property of the entire platelet plasma membrane or is a distinct membrane component released from activated platelets 10 remains unclear. The overall catalytic surface activity of activated platelets must include the contribution made by membrane-associated coagulation factors. In this regard, a platelet-associated factor V-like activity (platelet factor 1 or PF1) was first described in detail by Ware, Fahey, and Seegers over 30 years ag0.l Further