Signal transduction in the platelet activation induced by IgG anti-streptokinase and anisoylated plasminogen-streptokinase activator complex

Streptokinase (SK) is one of the plasminogen activators currently used in therapeutics. SK antibodies may appear in the blood after thrombolytic therapy with SK or after-hemolytic streptococci infection. Such antibodies may both activate platelets and neutralize the ability of SK to convert plasminogen into plasmin. We previously demonstrated that platelet activation induced by the combination of IgG anti-SK and anisoylated plasminogen-SK activator complex (APSAC) is mediated by Fc gamma RIIa1 receptor. However, the mechanism by which IgG anti-SK and APSAC (or SK) transduce an activating signal across the platelet plasma membrane remains unknown. We have demonstrated in the present study that the platelet aggregation induced by the combination of IgG anti-SK and APSAC is accompanied by an increase in inositol phosphate, Ca2+ mobilization and thromboxane (Tx) A2 generation. Neomycin, erbstatin and GF 109203X, which inhibit phospholipase C (PLC), protein tyrosine kinase (PTK) and protein kinase C (PKC) activities, respectively, abolished platelet aggregation induced by IgG anti-SK plus APSAC, indicating the pivotal roles of the PLC, PTK and PKC pathways in this immunological activation. In addition, TxA2 generation is also important since aspirin, a cyclooxygenase inhibitor and SQ 29548, a TxA2 receptor antagonist, showed significant inhibition of the platelet response. The contribution of released ADP was confirmed using apyrase, which significantly inhibited IgG anti-SK plus APSAC-induced platelet aggregation. Finally, WEB 2086, a platelet-activating factor (PAF) receptor antagonist, was not effective, indicating that PAF is not involved in this process. APSAC- or SKinduced platelet activation may limit the therapeutic effectiveness of the drug and may contribute to the pathogenesis of early reocclusion. The study of the mechanism leading to APSAC-induced platelet activation could be relevant for a better understanding of the physiopathology of immune complex disorder diseases and thrombolytic treatment failure.     

Original Article: Streptokinase Modifies in Vitro Platelet Aggregation by Two Mechanisms: Reduced Aggregation due to Fibrinogenolysis and Enhanced Aggregation via an Immunological Reaction

Both inhibition and enhancement of platelet aggregation have been observed after exposure to streptokinase (SK) in vitro. Recently we have shown that inhibition of aggregation appears to be related to the fraction containing the fibrinogen degradation product, fragment E. In addition, SK may initiate platelet aggregation by a mechanism involving specific anti-SK antibodies and plasminogen. Two monoclonal antibodies (MoAbs) (PL2-49 and LeoA1) were used to assess the immunological activation of platelets in SK-induced platelet aggregation and in SK-enhanced ADP-induced platelet aggregation. The anti-SK titers in healthy volunteers' and patients' (previously treated with SK for acute myocardial infarction) plasma, were measured using a one-site non-competitive ELISA. Serum from patients was used for the purification of IgG anti-SK by affinity chromatography. We confirmed that the degree of fibrinogen degradation is a major determinant of the aggregation inhibition induced by SK. SK-induced platelet aggregation and SK-enhanced ADP-induced platelet aggregation require the interaction of the Fc domain of the anti-SK antibodies with the FcyRII located on the platelet membrane, since they are blocked by the MoAb IV-3 directed against FcyRII. Classification of the subjects according to their responses to specific MoAbs (PL2-49 and LeoA1) supports the essential role played by immunological activation of platelets in SK-induced platelet aggregation and in SK-enhanced ADP-induced platelet aggregation. The ability of anti-SK antibodies to promote SK-induced platelet aggregation and SK-enhanced ADP-induced platelet aggregation, seems to result from the interaction between two separate mechanisms: the fist mechanism is based on immunological activation of platelets and the second is related to the intervention of a defined subset of anti-SK antibodies.     

Streptokinase-induced platelet aggregation. Prevalence and mechanism

BACKGROUND. Streptokinase (SK) is a bacteria-derived protein and one of the plasminogen activators that is currently available for therapeutic use. Exposure to SK induces synthesis of specific antibodies that may initiate platelet aggregation and paradoxical clot propagation during treatment. METHODS AND RESULTS. Using platelet-rich plasma (PRP), we found that SK (5,000 units/ml) but not urokinase (2,500 units/ml) or recombinant tissue-type plasminogen activator (2,500 units/ml) caused platelet aggregation in PRP from 14 of 100 normal volunteers. In 13 consecutive patients treated with SK for acute myocardial infarction, SK-mediated platelet aggregation was induced in five patients within 1 week after treatment. SK-mediated platelet aggregation was associated with significantly increased titers of both anti-SK antibodies and SK-neutralizing activity in plasma; it was partially inhibited by aspirin (1 mM) and by aprotinin (500 kallikrein inhibitor units/ml) and completely inhibited by tranexamic acid (1 mM) and by prostaglandin E1 (9 microM). Addition of SK (1,000 or 5,000 units/ml) induce a statistically significant dose-dependent thromboxane B2 release in mixtures of PRP with plasma from subjects with SK-induced aggregation but not in samples of PRP mixed with plasma from nonresponders; addition of recombinant tissue-type plasminogen activator (1 or 50 micrograms/ml) did not induce thromboxane B2 release. Mixing experiments with PRP and immunoglobulin G from reactive and nonreactive donors revealed that SK-induced aggregation requires the presence of anti-SK antibodies. When 125I-SK (50 nM) was used, platelets preincubated with plasminogen (0.5 microM) bound 9,500 +/- 600 (mean +/- SEM, n = 6) molecules SK/platelet, which increased to 25,000 +/- 3,100 molecules/platelet after thrombin stimulation. Tranexamic acid (1 mM) blocked specific binding of SK to resting platelets. CONCLUSIONS. These data demonstrate that SK-induced platelet aggregation is initiated by the binding of anti-SK antibodies to the SK-plasminogen complex located on the platelet surface. SK-induced platelet activation may limit the therapeutic effectiveness of the drug, and in view of the high prevalence of aggregation in a normal population, prospective evaluation of the effects of platelet aggregation during treatment with SK is warranted.     

Comparison of thrombolytic agents: selected hematologic, vascular and clinical events

Plasminogen activators of distinct structure and biochemical action seem to be more equivalent than unique regarding induced blood changes and clinical complications. All of the activators ultimately degrade substrate through plasmin, resulting in a striking hypocoagulable state characterized primarily by a decrease in fibrinogen concentration. Infusion regimens are inversely proportional to the half-life of the activator, which is relatively long with anisoylated plasminogen streptokinase activator complex (APSAC), intermediate for streptokinase (SK) and urokinase (UK), and very short for recombinant tissue plasminogen activator (rt-PA) and recombinant single-chain urokinase plasminogen activator (scu-PA). After therapy is discontinued, hypofibrinogenemia persists until activator is cleared from the blood, then is slowly corrected over 48 hours, regardless of which thrombolytic agent has been used. Coagulation and platelet activity may be transiently accentuated soon after administration of the agent. Hypercoagulability contributes to vascular reocclusion, especially when acting in concert with the thrombogenic influences of residual thrombus and the original ruptured atherosclerotic plaque. In the first 3 to 4 hours after symptom onset, coronary artery reperfusion can be achieved with all of the thrombolytic agents in 50 to 60% of patients, with a greater thrombolytic potential of rt-PA over SK in thrombi of greater than 4 hours' duration. After coronary artery reperfusion, reocclusion occurs in 10 to 20% of patients, more often after rt-PA than SK treatment. Antiplatelet agents such as aspirin decrease the incidence of reocclusion and when added to either SK or rt-PA, decrease mortality after acute myocardial infarction by half. APSAC appears to have a maximal beneficial effect in reducing mortality even without aspirin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Streptokinase-induced platelet activation involves antistreptokinase antibodies and cleavage of protease-activated receptor-1

Streptokinase activates platelets, limiting its effectiveness as a thrombolytic agent. The role of antistreptokinase antibodies and proteases in streptokinase-induced platelet activation was investigated. Streptokinase induced localization of human IgG to the platelet surface, platelet aggregation, and thromboxane A(2) production. These effects were inhibited by a monoclonal antibody to the platelet Fc receptor, IV.3. The platelet response to streptokinase was also blocked by an antibody directed against the cleavage site of the platelet thrombin receptor, protease-activated receptor-1 (PAR-1), but not by hirudin or an active site thrombin inhibitor, Ro46-6240. In plasma depleted of plasminogen, exogenous wild-type plasminogen, but not an inactive mutant protein, S(741)A plasminogen, supported platelet aggregation, suggesting that the protease cleaving PAR-1 was streptokinase-plasminogen. Streptokinase-plasminogen cleaved a synthetic peptide corresponding to PAR-1, resulting in generation of PAR-1 tethered ligand sequence and selectively reduced binding of a cleavage-sensitive PAR-1 antibody in intact cells. A combination of streptokinase, plasminogen, and antistreptokinase antibodies activated human erythroleukemic cells and was inhibited by pretreatment with IV.3 or pretreating the cells with the PAR-1 agonist SFLLRN, suggesting Fc receptor and PAR-1 interactions are necessary for cell activation in this system also. Streptokinase-induced platelet activation is dependent on both antistreptokinase-Fc receptor interactions and cleavage of PAR-1. (Blood. 2000;95:1301-1308)     

Intravenous thrombolytic therapy in myocardial infarction: An analytical review

The properties and physiological effects of three currently FDA-approved thrombolytic agents, streptokinase (SK), tissue plasminogen activator (tPA), and anisoylated plasmminogen activator complex (APSAC) are reviewed. All thrombolytic agents have been shown to reduce mortality postmyocardial infarction (MI). Comparative trials have failed to demonstrate a difference between the effects of tPA, SK, and APSAC on mortality. In addition, no consistent difference between the three agents on ejection fraction (EF) has been found despite a superior reperfusion rate with tPA at 90 min. Further-more, reinfarction and interventional procedure rates were significantly higher after thrombolytic treatment, and the incidence of total strokes was higher with tPA than SK in some comparative studies. Based on analysis of the published megatrials, SK is a more cost-effective thrombolytic agent for patients with acute MI than tPA or APSAC.     

New thrombolytic agents in myocardial infarction

Fibrinolysis is a physiological process which aims at dissolving intravascular thrombi and is mediated by activation of plasminogen to plasmin. Streptokinase (SK) and urokinase (UK) are non-specific plasminogen activators. They have proved effective as thrombolytic agents, but their use is limited by the risk of haemorrhages due to systemic fibrinogenolysis. More fibrin-specific drugs have recently been developed. One is a tissue plasminogen activator (t-PA), the other is a urokinase precursor (pro-UK), also called single chain urokinase plasminogen activator (scu-PA). Genetic engineering techniques have resulted in the large-scale production of a "recombinant t-PA" (rt-PA) and a "recombinant scu-PA" (r scu-PA) for therapeutic use, notably in acute myocardial infarction. In vitro, these two drugs exhibit a thrombolytic activity that is equal to, or greater than that of SK or UK. In vivo, their fibrinogenolytic effect is less pronounced, and their thrombolytic effect greater than those of SK or UK. "Acyl-enzymes" have more recently emerged. These are inactive acylated SK-plasminogen complexes which progressively become effective in plasma after deacylation. So far, the most extensively studied of these complexes is BRL 26921 (anisoylated plasminogen streptokinase activator complex, or APSAC) which is administered by bolus intravenous injection. It is more thrombolytic than SK but produces systemic fibrinogenolysis to an equivalent degree. Injected intravenously (by infusion or bolus) during the first hours of a coronary infarction these three new thrombolytic agents have proved effective in promoting coronary reperfusion, with an early coronary patency rate of 70-75%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reperfusion Adjunctive Therapy: Platelet Active Agents

Changes in a coronary artery thrombus resulting from platelet activation may affect the speed of thrombolysis and the frequency of reocdusion after thrombolytic therapy. In studies performed in canine models, we have found that selected thromboxane and serotonin receptor antagonists given in combination markedly enhance the thrombolytic effect of tissue plasminogen activator, probably by preventing further platelet activation and their incorporation into a thrombus during thrombolytic therapy. The combination of a thromboxane and serotonin receptor antagonist given with heparin and tissue plasminogen activator is effective in delaying or preventing reocdusion after discontinuation of tissue plasminogen activation in experimental canine models in whom coronary artery thrombosis has been induced by an indwelling copper coil.     

Pharmacokinetics, thrombolytic efficacy and hemorrhagic risk of different streptokinase regimens in heparin-treated acute myocardial infarction

The systemic activator activity of 4 streptokinase (SK) regimens (250,000 IU intracoronary, group A; 500,000 IU, group B; 1.5 X 10(6) IU, group C; and 30 U anisoylated plasminogen streptokinase activator complex (APSAC) intravenously, group D) was tested with the fibrin plate technique. One hour after initiation of treatment, the activator activity was highest after APSAC (3.6 +/- 0.9 U), slightly but not significantly less after SK 1.5 X 10(6) IU (3.0 +/- 0.7), and significantly less after SK 500,000 IU (1.6 +/- 0.5) and 250,000 IU (0.6 +/- 0.5), p less than 0.001. After SK, activator activity half-lives were 184 minutes (group B) and 169 minutes (group C), and after APSAC 188 minutes (group D). These were all in agreement with greater than 12 hour duration of changes in other markers of systemic fibrinolysis (euglobulin lysis time) and substrates depletion (fibrinogen, plasminogen, alpha 2 antiplasmin). In extended pilot clinical groups given identical thrombolytic regimens during full anticoagulation with heparin, angiographic coronary patency was found in 83% (35 of 42) after intracoronary SK (group 1), in 73 and 75%, respectively, after 500,000 IU (31 of 43) and 1.5 X 10(6) IU (30 of 40) (group 2 and 3, difference not significant) and 80% (8 of 10) after the 30-U bolus of APSAC (group 4). The overall hemorrhagic risk was 24%, equally distributed among the 4 regimens and mostly (91%) related to catheters. The incidence of bleeding unrelated to vessel puncture was 4%; no deaths occurred. It is concluded that APSAC is the most fibrinolytic regimen but its potential thrombolytic superiority over SK remains to be demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

The roles of alpha IIb beta 3-mediated outside-in signal transduction,thromboxane A2, and adenosine diphosphate in collagen-induced platelet aggregation

Collagen-induced activation of platelets in suspension leads to alpha(IIb)beta(3)-mediated outside-in signaling, granule release, thromboxane A2 (TxA2) production, and aggregation. Although much is known about collagen-induced platelet signaling, the roles of TxA2 production, adenosine diphosphate (ADP) and dense-granule secretion, and alpha(IIb)beta(3)-mediated outside-in signaling in this process are unclear. Here, we demonstrate that TxA2 and ADP are required for collagen-induced platelet activation in response to a low, but not a high, level of collagen and that alpha(IIb)beta(3)-mediated outside-in signaling is required, at least in part, for this TxA2 production and ADP secretion. A high level of collagen can activate platelets deficient in PLC gamma 2, G alpha q, or TxA2 receptors, as well as platelets treated with a protein kinase C inhibitor, Ro31-8220. Thus, activation of alpha(IIb)beta(3) in response to a high level of collagen does not require these signaling proteins. Furthermore, a high level of collagen can cause weak TxA2 and ADP-independent aggregation, but maximal aggregation induced by a high level of collagen requires TxA2 or secretion.     

Effects of streptokinase and recombinant tissue plasminogen activator on platelet aggregation in whole blood

Streptokinase (SK) frequently induced platelet aggregation when added to citrated whole blood in vitro, but aggregation did not occur in corresponding samples of platelet-rich plasma (PRP). The aggregation occurred at concentrations of SK that are achieved after systemic infusion and was significantly more extensive in blood from men than women. SK-induced aggregation was accompanied by TXB(2) formation and release of (14)C-5HT and was inhibited by aspirin, by sulotroban, a TXA(2) antagonist, and by apyrase, an enzyme which removed ADP from plasma. Aggregation was also inhibited by each of three agents that interrupt the fibrinolytic pathway: epsilon-aminocaproic acid (ACA), aprotinin and alpha-2-antiplasmin. It is suggested that the aggregation is consequent to platelet activation by plasmin formed on the platelet surface, with ADP from red cells playing a part. In contrast to results obtained in vitro, prior administration of SK to man resulted in inhibition of the aggregation that occurs when this agent is added to whole blood. This effect was reproduced in vitro by pre-incubating blood with SK prior to carrying out aggregation studies. Similar inhibition was obtained when plasmin was added to blood and it is suggested that this effect of SK may be mediated by plasmin formed in plasma. In contrast to the pro-aggregatory effects of SK in vitro, recombinant tissue plasminogen activator (rt-PA) only inhibited platelet aggregation in whole blood. rt-PA inhibited the aggregation induced by a wide range of agents suggesting a central mechanism of action. Inhibition of aggregation was prevented by ACA, suggesting that this is also mediated by plasmin formed in plasma. The relevance of these observations is discussed in relation to the increasing use of fibrinolytic therapy in acute thrombosis in man. It is suggested that slow infusion of SK should always be performed so as to maximise the inhibitory rather than potentiatory effect of this agent on platelet aggregation, and that SK should only be used after prior administration of an anti-platelet agent.     

Botrocetin/VWF-induced signaling through GPIb-IX-V produces TxA2 in an alphaIIbbeta3- and aggregation-independent manner

Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates a signaling cascade that causes alphaIIbbeta3 activation and platelet aggregation. Previous work demonstrated that botrocetin (bt)/VWF-mediated agglutination activates alphaIIbbeta3 and elicits adenosine triphosphate (ATP) secretion in a thromboxane A2 (TxA2)- and Ca2+-dependent manner. This agglutination-elicited TxA2 production occurs in the absence of ATP secretion. However, the signaling components and signaling network or pathway activated by GPIb-mediated agglutination to cause TxA2 production have not been identified. Therefore, the focus of this study was to elucidate at least part of the signal transduction network or pathway activated by GPIb-mediated agglutination to cause TxA2 production. The phosphatidylinositol 3-kinase (PI3K) selective inhibitor wortmannin, and mouse platelets deficient in Lyn, Src, Syk, Src homology 2 (SH2) domain-containing leukocyte protein 76 (SLP-76), phospholipase Cgamma2 (PLCgamma2), linker for activation of T cells (LAT), or Fc receptor gamma-chain (FcRgamma-chain) were used for these studies. LAT and FcRgamma-chain were found not to be required for agglutination-driven TxA2 production or activation of alphaIIbbeta3, but were required for granule secretion and aggregation. The results also clearly demonstrate that bt/VWF-mediated agglutination-induced TxA2 production is dependent on signaling apparently initiated by Lyn, enhanced by Src, and propagated through Syk, SLP-76, PI3K, PLCgamma2, and protein kinase C (PKC).     

Streptokinase inhibits acute platelet thrombus formation in stenosed dog coronary arteries

Previous evidence has suggested that plasmin, in addition to its proteolytic action on fibrin, may affect platelet function. To test the effects of plasmin generated in vivo by the thrombolytic agent streptokinase (SK) on platelet-dependent vascular occlusion, we have used a well-established canine model of experimental coronary artery stenosis which produces platelet aggregate-dependent cyclical variations in coronary blood flow. Infusion of SK into 22 dogs at doses sufficient to cause a systemic lytic state led to complete abolition of cyclical blood flow reductions (CFR's) at sites of coronary artery injury. Inhibition of coronary platelet occlusion was associated with marked prolongations of the bleeding time (from 3.2 ± 0.6 min before to 14 ± 5 min after SK infusion, mean ± SD, n = 22). Despite the striking effects of SK on in vivo platelet-vessel wall interactions, only platelet aggregation in response to collagen was diminished among the ex vivo parameters of platelet function that were studied simultaneously. Platelet aggregation in response to other agonists, thromboxane A2 production, monoclonal antibody binding to platelet membrane glycoprotein (Gp) IIb-IIIa, Gp Ib-dependent botrocetin-induced platelet aggregation and platelet levels of cyclic AMP were not significantly altered. Therefore the thrombolytic agent streptokinase appears to cause important inhibitory effects on in vivo platelet reactivity with injured vascular intimal surfaces, possibly due to localised changes in platelet aggregate formation in the microenvironment of exposed collagen. These findings suggest that plasmin generated by thrombolytic agents may exhibit platelet inhibitory activity, and that this effect may be important in reestablishing blood flow in certain forms of platelet-mediated arterial thromboses.     

Human platelets activation by convulxin is accompanied by tyrosyl-phosphorylation of PLCgamma2 and occurs independently of integrin alphaIIbbeta3

In the present report we show that convulxin (Cvx), a C-type lectin from Crotalus durissus terrificus venom, induces platelet agregation and phospholipase C (PLC) activation by a protein tyrosine kinase (PTK)-dependent pathway. In addition, Cvx stimulates a rapid increase in tyrosine phosphorylation of human platelet proteins with molecular masses of 40, 72/74, 78/80 and 120 kDa, followed by dephosphorylation of some proteins. However, platelet aggregation was accompanied by the phosphorylation of a 105-kDa molecular mass protein. Furthermore, Cvx stimulates a rapid-tyrosyl phosphorylation of a 145-kDa protein that was identified as PLC gamma 2. Protein tyrose phosphatase (PTP) induced by Cvx was not blocked when platelets were stimulated in the presence of indomethacin, apyrase, EDTA or RGDS peptide, but inhibited by staurosporine and genistein. These results indicate that PTP is chronologically proximal to Cvx binding to platelets, and it is independent of platelet aggregation or fibrinogen binding to integrin alphaIIbbeta3. On the other hand, the phosphorylation step, and the phosphorylation of the 105-kDa protein, were both inhibited by RGDS and EDTA, which suggests that the integrin alphaIIbbeta3 beta is involved in these steps. Our results, taken together, show that Cvx induces platelet IIb 3 aggregation in a similar manner as collagen and collagen-related peptides that also trigger platelet aggregation by a PTK-dependent pathway, and stimulate tyrosyl-phosphorylation of PLC gamma 2. However, Cvx is unique among platelet receptor agonists, because under test-tube stirring conditions it induces a PTP profile independently of integrin alphaIIbbeta3.     

Effects of P2Y 1 and P2Y 12 receptor antagonists on platelet aggregation induced by different agonists in human whole blood

ADP plays a major role in the amplification of platelet aggregation induced by other platelet agonists. ADP initiates platelet activation via the P2Y 1 receptor and amplifies platelet activation via the P2Y 12 receptor. Using the selective P2Y 1 receptor antagonist A2P5P and the selective P2Y 12 receptor antagonist AR-C69931MX, we assessed the relative contributions of P2Y 1 receptor and P2Y 12 receptor activation to platelet aggregation in hirudin-anticoagulated whole blood induced by PAF, 5HT, epinephrine, TRAP, streptokinase, U46619 and collagen. The effects of aspirin were assessed concurrently. A2P5P and AR-C69931MX variably inhibited aggregation induced by most of the agonists studied, whereas aspirin only inhibited aggregation induced by streptokinase and collagen. In some experiments, A2P5P and AR-C69931MX yielded additive inhibition of aggregation. All three antagonists interacted synergistically to inhibit collagen-induced aggregation. These studies demonstrate that P2Y 1 receptor activation plays a significant role in amplifying aggregation induced by agonists other than ADP, in addition to the established roles of P2Y 12 receptor activation and thromboxane A 2 synthesis.     

The Ideal Thrombolytic Agent: GISSI-2 and ISIS-3

Streptokinase (SK) and tissue plasminogen activator (alteplase [tPA]) are by far the most intensively studied thrombolytic agents, but only recently a direct head-to-head comparison (GISSI-2 Trial, in which 12,490 patients have been randomized) has become available to the scientific community. This trial, with its international extension (the International tPA/SK Mortality Trial) recruited more than 20,000 patients, showed that the two drugs tested (SK and tPA) were substantially comparable as far as the clinical events were considered. ISIS-3, the largest randomized clinical trial ever performed, compares SK, tPA, and APSAC in a factorial design. Its results will be available before spring, 1991. It is likely that these two studies, if the results will be consistent, will give a definitive answer about comparison among thrombolytic drugs. As a consequence of that achievement, the interest of the scientific community will possibly be directed towards the long-term prevention of the infarct complications, namely ventricular remodeling and infarct expansion or life-threatening arrhythmias.     

Equine peritoneal macrophage production of thromboxane and prostacyclin in response to platelet activating factor and its receptor antagonist SRI 63-441

The formation of eicosanoids may be a primary route through which platelet activating factor (PAF) exerts its effects during endotoxemia. Since endotoxemia is a common cause of death in horses, a study was conducted to determine whether PAF could stimulate equine macrophage release of thromboxane A2 (TxA2) and prostacyclin (PGI2) and whether a PAF-receptor antagonist would alter macrophage eicosanoid synthesis. Equine peritoneal macrophages were cultured from clinically normal horses and exposed to various concentrations of PAF, the PAF-receptor antagonist SRI 63-441, endotoxin, or a combination of these. The supernatant concentrations of TxB2 and 6-keto-prostaglandin F1 alpha were determined after 6 hr incubation. The media concentration of TxB2 was increased significantly above baseline after treatment of macrophages with PAF (10(-7) to 10(-5) M), and the magnitude was similar to that induced by endotoxin. This TxB2 increase was not prevented by prior treatment of macrophages with SRI 63-441. SRI 63-441 (greater than or equal to 5 x 10(-5) M) significantly enhanced macrophage TxA2 synthesis, as well as its production of PGI2, similar to the effects of endotoxin. Media concentrations of 6-keto-prostaglandin F1 alpha were not increased significantly above baseline after treatment of macrophages with PAF (10(-8) to 10(-5) M). These results suggest that PAF may cause increased TxA2 release during endotoxemia, which may not be preventable by use of the PAF-receptor antagonist SRI-63-441, which is capable of inhibiting PAF-induced aggregation of equine platelets.     

Thrombolysis for Acute Myocardial Infarction: Making Sense of The Clinical Trials Data

Trials during the 1980s established that thrombolytic therapy restores coronary blood flow rapidly and reduces mortality compared to control or placebo therapy in acute myocardial infarction. Comparative studies have generated controversy, however, in that the newer agents tissue plasminogen activator (tPA) and anistreplase (APSAC), shown to open arteries more rapidly than streptokinase (SK), achieve no greater mortality reductions. In these trials, tPA may have been disadvantaged because adjunctive IV heparin therapy was not given. This possibility is being explored in the Global Utilization of Streptokinase and tPA for Occluded Coronary Arteries (GUSTO) study. However, heparin may not entirely explain the discrepancy because (1) among patients in International Studies of Infarct Survival (ISIS-3) given IV heparin out of protocol, no difference in mortality between tPA and SK was observed; (2) reinfarction was actually slightly less after tPA; and (3) APSAC, longer acting and more efficient than SK and not requiring early IV heparin, achieved no greater mortality reductions. Another explanation for the discrepancy between 90-minute patency and mortality effects among agents is that benefit in the usual time frame of treatment (2–6 hours after onset of symptoms) depends more on diastolic effects (improved healing with preserved diastolic size, shape) than on systolic effects (reduction in infarct size) and is less time-dependent. A similar survival outcome is then consistent with the observation that all three agents lead to similar plateau patency rates at ≥ 3 hours. Prehospital throtnbolysis trials are exploring the possibility that greater functional and mortality benefits are associated with therapy given very early (within 1–2 hours). Differential benefits might be seen when regimens with differing thrombolytic efficiencies are given within 1–2 hours of symptom onset, but this possibility must be established by direct comparative trials. Future trials should emphasize improved myocardial salvage by more rapid delivery of thrombolytic therapy as well as the use of more efficient thrombolytic regimens.     

In vitro effect of plasmin on human platelet function in plasma. Inhibition of aggregation caused by fibrinogenolysis.

BACKGROUND. Plasmin has been reported both to activate platelets and to inhibit platelet functions. The latter effect was thought to be caused by proteolysis of the main membrane glycoproteins. METHODS AND RESULTS. We found that incubation of citrated human platelet-rich plasma with streptokinase (SK) (300 IU/ml) does not produce any detectable activation but leads to a time-dependent inhibition of ADP-induced aggregation accompanied by substantial fibrinogenolysis. These effects were abrogated by previous addition of a plasmin inhibitor, aprotinin. Crossover experiments (SK-treated or control platelets mixed with SK-treated or control plasma) demonstrated that the platelets remained functional and that the aggregation defect was caused by fibrinogenolysis. Further experiments (addition of purified fibrinogen to fibrinogen-depleted plasma with either SK or thrombin) suggested that in addition to the low residual level of fibrinogen, fibrinogen degradation products had an inhibitory effect. Under the same conditions, tissue-type plasminogen activator (t-PA) (3,000 ng/ml) had no effect on platelet aggregation, and plasma fibrinogen was not significantly lowered. The effects on glycoproteins IIb-IIIa of incubation with SK, t-PA, or plasmin were assessed with immunoblots with murine monoclonal antibodies directed against either part of the complex, which is the receptor for fibrinogen. Proteolysis was detected only in the presence of EDTA, a potent chelator of divalent cations. CONCLUSIONS. The incubation of human platelets in citrated plasma with SK concentrations obtained during therapy leads to an aggregation defect that is related to the decrease in fibrinogen, the adhesive protein involved in this function, and to the impeding effect of fibrinogen degradation products on its binding onto platelets but not to an alteration of the corresponding platelet receptor, the heterodimer glycoproteins IIb-IIIa.     

Abnormal inside-out signal transduction-dependent activation of glycoprotein IIb-IIIa in a patient with impaired pleckstrin phosphorylation

Platelet-agonist interaction results in activation of glycoprotein (GP) IIb-IIIa complex and fibrinogen binding, a prerequisite for platelet aggregation. Fibrinogen binding exposes new antibody binding sites on GPIIb-IIIa (ligand-induced binding sites: LIBS). Signal transduction events, including pleckstrin phosphorylation by protein kinase C (PKC), are considered to regulate GPIIb-IIIa activation. We studied a 16-year- old white male with lifelong mucocutaneous bleeding manifestations and abnormal platelet aggregation and secretion in response to multiple agonists. Pleckstrin phosphorylation was diminished in response to platelet-activating factor (PAF; 4 and 400 nmol/L) and thrombin (0.05 U/mL). Binding of monoclonal antibodies (MoAbs) 10E5 and A2A9, which bind to both resting and activated GPIIb-IIIa, was normal. Binding of MoAb PAC1, which binds to only activated GPIIb-IIIa, was diminished upon activation with PAF, adenosine diphosphate (ADP), thrombin receptor agonist peptide (SFLLRN), A23187, and 1,2-dioctonylglycerol (DiC8). Signal transduction-dependent LIBS expression (studied using MoAb 62) induced by ADP, SFLLRN, and DiC8 and signal transduction- independent LIBS expression induced by RGDS peptide or disintegrin albolabrin were normal or minimally decreased, indicating the presence of intact ligand binding sites. We conclude that the patient's platelets have a defect in inside-out signal transduction-dependent GPIIb-IIIa activation due to an upstream defect in the signal transduction mechanisms rather than in the GPIIb-IIIa complex itself. Our findings extend the spectrum of congenital mechanisms leading to impaired aggregation from defects in GPIIb-IIIa per se to aberrations in signaling mechanisms.