Support Platelet Aggregation Research Articles

Influence of alpha-adrenergic blockade on platelet-mediated thrombosis in stenosed canine coronary arteries.

The functional significance of platelet alpha-adrenergic receptors in vivo is uncertain. The aim of this study was to elucidate their role in experimental coronary thrombosis. In 46 open-chest dogs with a critical coronary stenosis produced by plicating the coronary artery wall with a suture, blood flow showed cyclical reductions followed by an abrupt return to control levels. The flow reduction were previously shown to be caused by platelet aggregation. In the present study they were unaffected by heparin (1,000 U . kg-1 . h-1) at the start of the experiment but consistently abolished by aspirin (30 mg . kg-1) at the end of the protocol, further supporting platelet aggregation as the primary mechanism. After 1 h of observation, dogs were assigned to one of the following groups: control (no intervention, n = 9), phentolamine (a nonselective alpha-blocker) "low dose" (0.07 mg . kg-1 bolus followed by 0.1 mg . kg-1 . min-1, n = 4), phentolamine "high-dose" (3 mg . kg-1 bolus followed by 0.07 mg . kg-1 . min-1, n = 6), prazosin (an alpha 1-blocker, 2 mg . kg-1, n = 8), yohimbine (an alpha 2-blocker, 3 mg . kg-1, n = 11) or prazosin + yohimbine (same doses, n = 8). In controls, cyclical reductions in flow continued unchanged for another hour. Phentolamine effected a partial dose-related inhibition of flow reductions; however, prazosin and yohimbine, given separately or in combination, failed to produce any significant effect despite an alpha-blocking action equivalent to or greater than that of phentolamine (alpha-agonist dose-response studies).(ABSTRACT TRUNCATED AT 250 WORDS)

Asialo von Willebrand factor interactions with platelets. Interdependence of glycoproteins Ib and IIb/IIIa for binding and aggregation.

Asialo von Willebrand factor (AS-vWf) binds to and aggregates normal human platelets in the absence of ristocetin. Maximal specific binding of AS-vWf is 1-2 micrograms vWf protein/10(8) platelets. Despite the specificity of the binding, only 60% of the bound AS-vWf can be dissociated after equilibrium has been reached. We investigated the site of binding and the mechanism of aggregation of platelets by AS-vWf by (a) pre-incubating platelets with either of two monoclonal antibodies, one against glycoprotein Ib (GPIb) or a second against the glycoprotein IIb/IIIa complex (GPIIb/IIIa), and (b) varying the concentration of fibrinogen in the medium. The results of our studies indicate that AS-vWf binds initially to GPIb. This binding then results in the exposure of receptors for AS-vWf on GPIIb/IIIa. In the presence of plasma fibrinogen, both AS-vWf and fibrinogen bind to GPIIb/IIIa. In the presence of plasma fibrinogen, 50% more AS-vWf binds to the platelet, and this additional AS-vWf binds almost exclusively to GPIIb/IIIa. Despite this enhanced binding of AS-vWf in the absence of fibrinogen, platelet aggregation is much less than that which occurs in the presence of plasma fibrinogen. Comparative studies of AS-vWf binding to normal platelets and the platelets of patients with Glanzmann's thrombasthenia reveal decreased binding to the thrombasthenic platelets and a marked decrease in the extent of platelet aggregation. These studies indicate that AS-vWf binding to, and ensuing aggregation of, platelets is different from that observed with intact vWf protein when platelets are stimulated with either ristocetin or thrombin. The AS-vWf binds to GPIb which, in turn, makes additional AS-vWf receptors available on GPIIb/IIIa. If plasma fibrinogen is present, it competes with the AS-vWf for binding to GPIIb/IIIa and causes aggregation of platelets. In the presence of plasma fibrinogen, more of the AS-vWf binds to GPIIb/IIIa, but this AS-vWf is much less effective than fibrinogen in supporting platelet aggregation.

A regimen for low-dose aspirin?

The effects of different regimens of 40 mg aspirin on platelet thromboxane A2 synthesis and vascular prostacyclin synthesis were determined in patients who were undergoing elective surgery for removal of varicose veins. Aspirin 40 mg taken at intervals of 48 hours consistently reduced platelet thromboxane A2 synthesis to a level at which it failed to support platelet aggregation and the associated release reaction. This effect lasted for at least 36 hours. In contrast, aspirin 40 mg every 72 hours did not have the same consistent effect. Both dose regimens led to a reduction in vascular prostacyclin synthesis 12 hours after the last dose, but 36 or 72 hours after the last dose prostacyclin synthesis was not reduced; thus the inhibition of prostacyclin synthesis was short lived. If the balance between platelet thromboxane A2 and vascular prostacyclin synthesis is important in thrombosis 40 mg aspirin every 48 hours may have the maximum antithrombotic effect.

Identification of the fibrinogen receptor on human platelets by photoaffinity labeling.

Fibrinogen binding to receptors on stimulated platelets is a prerequisite for platelet aggregation. In order to identify the platelet fibrinogen receptor, we modified fibrinogen with the photoreactive, heterobifunctional cross-linking reagent methyl 4-azidobenzoimidate (MABI). MABI-fibrinogen was fully clottable and able to support platelet aggregation. To photoaffinity label the fibrinogen receptor, gel-filtered human platelets were incubated at 37 degrees C in the dark with 200 micrograms/ml of MABI-fibrinogen, 10 microM ADP, and 0.5 mM calcium. Irradiation of these platelets with ultraviolet light resulted in the incorporation of MABI-fibrinogen into the platelet surface. Incorporation could be prevented by excess native fibrinogen suggesting that MABI-fibrinogen had interacted with the fibrinogen receptor before photolysis. Examination of the irradiated platelets by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the photoactivated MABI-fibrinogen had been incorporated into a 105,000 molecular weight membrane polypeptide that also contained the PlA1 antigen. Thus, this polypeptide has the characteristics of the membrane glycoprotein IIIa. Previous studies have shown that thrombasthenic platelets lack this glycoprotein and fail to bind fibrinogen after stimulation by ADP. Consequently, our data suggest that glycoprotein IIIa constitutes at least one component of the platelet fibrinogen receptor.

DIFFERENTIAL INHIBITION BY LOW-DOSE ASPIRIN OF HUMAN VENOUS PROSTACYCLIN SYNTHESIS AND PLATELET THROMBOXANE SYNTHESIS

The capacity of venous tissue for prostacyclin synthesis was determined in 68 patients undergoing surgery for removal of varicose veins. A single dose of aspirin (81 mg or 300 mg) taken 14 h preoperatively strongly inhibited its synthesis, and the effect of 300 mg was still evident 48 h after ingestion. A single dose of 40 mg aspirin taken 14 h preoperatively had no effect on prostacyclin synthesis. The capacity of blood platelets to synthesise thromboxane (measured as malondialdehyde) was determined in volunteers before and at various times after ingestion of 300 mg or 40 mg aspirin. Both doses had an inhibitory effect that lasted for at least 96 h. The length of time for which the amount of thromboxane synthesised was insufficient to support platelet aggregation and the platelet release reaction depended on both the donor and the dose of aspirin. If prostacyclin and thromboxane are important in the pathogenesis of thrombosis, then doses of aspirin much lower than those used previously should be tested. The long-lasting effect of 300 mg aspirin on both venous tissue and platelets indicates that this dose is unlikely to produce a favourable prostacyclin/thromboxane balance.

Fibrinogen Receptor Exposure and Aggregation of Human Blood Platelets Produced by ADP and Chilling

AbstractADP-induced platelet aggregation is associated with the specific binding of fibrinogen to receptors. Since fibrinogen is also a cofactor for the spontaneous aggregation of chilled human platelets during rewarming, we examined the ability of these platelets to bind purified 126l-labeled fibrinogen. Specific binding similar to ADP-induced binding at room temperature was observed after the platelets had been chilled for 20 min at 0–2°C. Aggregation, however, occurred only when the ambient temperature was increased and was followed within 15–30 sec by loss of most of the bound fibrinogen and disaggregation. ADP- and cold-induced fibrinogen binding and aggregation were inhibited by 0.3 µM PGE“ 1 mM dibutyryl cyclic AMP, and 2.5 mM EDTA, unaffected by 208 µM colchicine, and absent from thrombasthenic platelets. Platelets change shape from discs to spiny spheres when they are exposed to ADP or cold. PGE1 and dibutyryl cyclic AMP prevented the shape change induced by ADP but not by chilling. Shape change, fibrinogen binding, and aggregation induced by ADP. but not by chilling, were inhibited by incubating platelets with 7.3 µM antimycin A and 4.9 mM 2-deoxy-D-glucose. Fixed platelets bound fibrinogen only if they were exposed to ADP before fixation but they failed to aggregate. Studies with fixed platelets showed that EDTA and a low pH prevent the interaction of fibrinogen with exposed receptors, whereas platelets stimulated with ADP in the absence of divalent cations or at low pH prior to fixation bound fibrinogen at physiologic pH, and this binding was inhibited by EDTA. The present studies provide the following clues about the requirements for fibrinogen receptor exposure and platelet aggregation. (1) ADP and chilling activate platelets by exposing fibrinogen receptors via a common pathway that is blocked by elevating cyclic AMP. (2) Chilling can bypass the seemingly ATP-dependent step for ADP-induced fibrinogen receptor exposure. (3) Receptor exposure by both ADP and chilling is independent of platelet shape. (4) Increased cyclic AMP inhibits the shape change induced by ADP but not that caused by chilling. (5) EDTA and low pH prevent aggregation by interfering with the binding of fibrinogen to exposed receptors. (6) Receptor mobility may be as important as fibrinogen binding in supporting platelet aggregation.

Fibrinogen receptor exposure and aggregation of human blood platelets produced by ADP and chilling

Abstract ADP-induced platelet aggregation is associated with the specific binding of fibrinogen to receptors. Since fibrinogen is also a cofactor for the spontaneous aggregation of chilled human platelets during rewarming, we examined the ability of these platelets to bind purified 126l-labeled fibrinogen. Specific binding similar to ADP-induced binding at room temperature was observed after the platelets had been chilled for 20 min at 0–2°C. Aggregation, however, occurred only when the ambient temperature was increased and was followed within 15–30 sec by loss of most of the bound fibrinogen and disaggregation. ADP- and cold-induced fibrinogen binding and aggregation were inhibited by 0.3 µM PGE“ 1 mM dibutyryl cyclic AMP, and 2.5 mM EDTA, unaffected by 208 µM colchicine, and absent from thrombasthenic platelets. Platelets change shape from discs to spiny spheres when they are exposed to ADP or cold. PGE1 and dibutyryl cyclic AMP prevented the shape change induced by ADP but not by chilling. Shape change, fibrinogen binding, and aggregation induced by ADP. but not by chilling, were inhibited by incubating platelets with 7.3 µM antimycin A and 4.9 mM 2-deoxy-D-glucose. Fixed platelets bound fibrinogen only if they were exposed to ADP before fixation but they failed to aggregate. Studies with fixed platelets showed that EDTA and a low pH prevent the interaction of fibrinogen with exposed receptors, whereas platelets stimulated with ADP in the absence of divalent cations or at low pH prior to fixation bound fibrinogen at physiologic pH, and this binding was inhibited by EDTA. The present studies provide the following clues about the requirements for fibrinogen receptor exposure and platelet aggregation. (1) ADP and chilling activate platelets by exposing fibrinogen receptors via a common pathway that is blocked by elevating cyclic AMP. (2) Chilling can bypass the seemingly ATP-dependent step for ADP-induced fibrinogen receptor exposure. (3) Receptor exposure by both ADP and chilling is independent of platelet shape. (4) Increased cyclic AMP inhibits the shape change induced by ADP but not that caused by chilling. (5) EDTA and low pH prevent aggregation by interfering with the binding of fibrinogen to exposed receptors. (6) Receptor mobility may be as important as fibrinogen binding in supporting platelet aggregation.

Support of Ristocetin-Induced Platelet Aggregation by Procoagulant-Inactive and Plasmin-Cleaved Forms of Human Factor VIII/von Willebrand Factor

Human factor VIII/von Willebrand factor (fVIII/vWf) was purified to homogeneity as defined by electrophoretic and immunologic criteria and tested for fVIII procoagulant and ristocetin cofactor activities. As little as 0.4 μg/ml of purified fVIII/vWf fully aggregated washed human platelets in the presence of ristocetin. Purified fVIII/vWf, whether thrombin-activated or -inactivated, and fVIII/vWf with its procoagulant activity abolished by a human inhibitor, supported ristocetin-induced platelet aggregation as effectively as native fVIII/vWf. Similarly, purified hemophilic fVIII/vWf protein and fVIII/vWf-like protein isolated from normal serum supported the aggregation of platelets in the presence of ristocetin. FVIII/vWf extensively degraded by human plasmin and exposed to denaturing solvents also supported platelet aggregation in the presence of ristocetin. Plasmin hydrolyzed fVIII/vWf to yield noncovalently bonded fragments that could be separated into two pools by gel filtration in guanidine hydrochloride. After dialysis into dilute neutral buffers, the pool of high molecular weight fragments supported significant ristocetin-induced platelet aggregation and cross-reacted with an antibody to native fVIII/vWf. Hence, unlike fVIII procoagulant activity, the ristocetin cofactor activity and antigenicity of fVIII/vWf are retained, despite changes in the structure and conformation of the molecule. These results suggest caution about the interpretation of procoagulant activity:fVIII/vWf antigen ratios or procoagulant activity:ristocetin cofactor activity ratios in whole plasma, fVIII/vWf concentrates or purified preparations of fVIII/vWf, since small amounts of minimally degraded, procoagulant-inactive fVIII/vWf cound markedly alter such values.

Asialofibrinogen supports platelet aggregation and adhesion to glass

Asialofibrinogen supports platelet aggregation and adhesion to glass

Asialofibrinogen Supports Platelet Aggregation and Adhesion to Glass

Fibrinogen is required for the initial phase of platelet aggregation induced by ADP and epinephrine and the adhesion of platelets to glass. Since the carbohydrate moiety of glycoproteins has been implicated in their interactions with cell surfaces, I investigated the role of fibrinogen's sialic acid in these platelet cofactor functions. Plasminogen-free human fibrinogen (which had no detectable factor VIII/von Willebrand factor antigen by electroimmunoassay) was treated with 0.1 U/ml CI. perfringens neuraminidase at pH 6.2 for 3 hr at 37°C and then further purified by gel filtration and glycine precipitation. The neuraminidase had no detectable protease activity against casein, with a lower limit of sensitivity of 20 mU trypsin/U neuraminidase. Desialylation was greater than 95 % complete, since the treated fibrinogen had

The role of vitreous collagen in platelet aggregation in vitro and in vivo.

Abstract The reaction between platelets and the macromolecular constituents present in connective tissue was studied using the vitreous as a model system. Direct injection of platelets into the rabbit vitreous resulted in the prompt formation of a membrane. In vitro aggregation experiments were performed using washed human platelets and fractions obtained from the bovine, rabbit, and owl monkey vitreous. In all three species only those fractions containing hydroxylysine and hydroxyproline supported platelet aggregation. Amino acid and carbohydrate analysis, as well as electron microscopic examination indicated that the guanidine-insoluble fraction from the bovine vitreous was composed only of collagen and that this appeared to be the constituent which caused platelet aggregation in vivo. The mechanism of platelet aggregation thus appears to be similar in vitro and in vivo. Support for this conclusion was obtained by analysis of the vitreous membrane formed in vivo which showed the presence of amino acids characteristic of collagen as well as an unidentified component also present in platelets. In experiments performed with platelets whose function had been modified by treatment with aspirin, membrane formation was inhibited in vivo. This finding indicated that adhesion to collagen is an essential prerequisite for aggregation. In vivo membrane formation occurred with less than 10 μg of collagen per milliliter and was not impaired by either its association with other structural proteins or the unusual organization of the fibers.