A micromethod to measure platelet aggregation and atp release by impedance.

A standard electrode has been modified in order to detect platelet aggregation by impedance in small volumes of whole blood. The modified electrode was constructed to fit into a standard 0.5-ml cuvette normally employed for detecting platelet aggregation in platelet-rich plasma (PRP) on a Chrono-log Aggregometer (Haverford, PA). Whole blood samples were mixed 1:1 with saline and total volumes of 1 ml, 500 microl and 300 microl were tested. ATP release was recorded concurrently with aggregation by measuring the luminescence from the firefly luciferin-luciferase reaction. The modified electrode could detect aggregation in the 300- and 500-microl samples with the same efficiency as the 1-ml samples. ATP release in the 300- and 500-microl samples yielded equivalent levels of release as detected with the 1-ml samples. This electrode is well suited for evaluation of platelet function in research and clinical blood samples where only small volumes of blood are available.

Unique pathway of thrombin-induced platelet aggregation mediated by glycoprotein Ib.

Thrombin plays a central role in normal and abnormal hemostatic processes. It is assumed that alpha-thrombin activates platelets by hydrolyzing the protease-activated receptor (PAR)-1, thereby exposing a new N-terminal sequence, a tethered ligand, which initiates a cascade of molecular reactions leading to thrombus formation. This process involves cross-linking of adjacent platelets mediated by the interaction of activated glycoprotein (GP) IIb/IIIa with distinct amino acid sequences, LGGAKQAGDV and/or RGD, at each end of dimeric fibrinogen molecules. We demonstrate here the existence of a second alpha-thrombin-induced platelet-activating pathway, dependent on GP Ib, which does not require hydrolysis of a substrate receptor, utilizes polymerizing fibrin instead of fibrinogen, and can be inhibited by the Fab fragment of the monoclonal antibody LJIb-10 bound to the GP Ib thrombin-binding site or by the cobra venom metalloproteinase, mocarhagin, that hydrolyzes the extracellular portion of GP Ib. This alternative alpha-thrombin pathway is observed when PAR-1 or GP IIb/IIIa is inhibited. The recognition sites involved in the cross-linking of polymerizing fibrin and surface integrins via the GP Ib pathway are different from those associated with fibrinogen. This pathway is insensitive to RGDS and anti-GP IIb/IIIa antibodies but reactive with a mutant fibrinogen, gamma407, with a deletion of the gamma-chain sequence, AGDV. The reaction is not due to simple trapping of platelets by the fibrin clot, since ligand binding, signal transduction, and second messenger formation are required. The GP Ib pathway is accompanied by mobilization of internal calcium and the platelet release reaction. This latter aspect is not observed with ristocetin-induced GP Ib-von Willebrand factor agglutination nor with GP Ib-von Willebrand factor-polymerizing fibrin trapping of platelets. Human platelets also respond to gamma-thrombin, an autoproteolytic product of alpha-thrombin, through PAR-4. Co-activation of the GP Ib, PAR-1, and PAR-4 pathways elicit synergistic responses. The presence of the GP Ib pathway may explain why anti-alpha-thrombin/anti-platelet regimens fail to completely abrogate thrombosis/restenosis in the cardiac patient.

Influence of vortex speed on fresh versus stored platelet aggregation in the absence and presence of extracellular ATP.

Platelets are subjected to vastly differing shear forces under laminar and nonlaminar flow patterns throughout the tortuous cardiovascular system. Different activation pathways appear to be associated with platelet adhesion and aggregation under high shear rates vs. low shear rates. We found that platelets continue to aggregate at very low stirring rates (100 RPM) and low shear forces although significantly less than at high stirring rates (1000 RPM). These conditions may model vortices encountered in vivo, such as downstream of partially occluded blood vessels. The extent of agonist-induced platelet aggregation, at varying stir rates, remained essentially unchanged between 1200 and 600 RPM. This was true for both freshly prepared and stored platelets even though the extent of aggregation was significantly reduced with stored platelets. Agonists used were thrombin, thrombin receptor activating peptide (TRAP), SFLLRNP, the thromboxane A2 mimetic, U46619, plus epinephrine and ADP+epinephrine. At lower stir rates (100-400 RPM), little or no difference in aggregation levels was observed between fresh and stored platelets, depending upon agonist used. This may indicate that old and young platelets, in vivo, would be equally active at vessel walls exposed to blood flowing through a slow vortex at low shear rates. ATP, released from activated platelets, may act as a potent regulator of platelet aggregation within a vortex where the resident time of platelets and bioactive molecules is greater than in laminar flow regions. High levels of extracellular ATP (100 microM) inhibited agonist-induced aggregation of fresh platelets to a greater extent than stored platelets, except with ADP+epinephrine where the converse was observed. Inhibition, in general, appeared to be inversely related to stir rates. Low levels of extracellular ATP (10 nM, 1 microM) generally stimulated agonist-induced aggregations independent of stir rates and to a greater extent with stored platelets than fresh platelets. Unraveling how hemostasis functions within microenvironments may facilitate ways to further regulate this process.

Permanent lesions of stored platelets correlate to pH and cell count while reversible lesions do not.

The demand for stored platelet concentrates (PC) for therapeutic transfusions has been increasing for the past three decades. Loss of platelet functionality increases with the length of storage time due to a multitude of factors collectively referred to as a platelet storage lesion. As more of the causes of the storage lesion have been defined, storage conditions have improved along with the therapeutic value of the transfused platelet samples. This report addressed new aspects of the storage lesion correlated with the pH of the storage medium. Platelet function was evaluated as aggregation induced by the synergistic agonist pair, U46619 (TXA2 mimetic) plus epinephrine or the strong agonists SFLLRNP (a peptide thrombin receptor agonist) or thrombin each added alone. Stored PC were compared to freshly prepared platelets as platelet-rich plasma (PRP) or washed platelets re-suspended in hepes Tyrode's buffer. The pH of the storage plasma, was inversely proportional to the cell count with platelets stored for 6 days. Agonist-induced platelet aggregation was significantly impaired by storage for 6-7 days as PRP; however, upon washing, a significant level of activity was restored. Washed platelets more accurately reflect conditions of transfused platelets that may regain activity in vivo. There appeared to be two subpopulations of stored PRP samples--one that retained activity and one that lost virtually all activity with the agonists tested. However, the lack of response to agonist observed with one subpopulation was reversed to the same level obtained with the active subpopulation upon washing. The subpopulation of stored PRP samples that were inactive with U46619-plus-epinephrine did not correspond to the subpopulation of samples that were nonresponsive to SFLLRNP, indicating that loss of activity with selected samples was possibly receptor specific. Loss of agonist-induced aggregation with PRP samples did not correlate with storage pH; however, the level of aggregation with washed platelets correlated significantly with pH. Results implied that pH caused a permanent storage lesion that could only be detected with washed platelets. A partially reversible lesion was superimposed on the pH lesion and was only detectable with PRP samples. Results indicate that continued attention must be paid to regulate the pH of stored PC even in the second generation plastic bags.

Cytokine-mediated erythroid maturation in megakaryoblastic human cell line HU-3.

HU-3 is a bipotential cell line derived from the bone marrow of a patient with megakaryoblastic leukemia. Continuously proliferating cells evolved from cultures supplemented with nutrient medium containing human serum and granulocyte-macrophage (GM) colony-stimulating factor (CSF). Growth and viability of the HU-3 cell line was strictly dependent on the presence of GM-CSF, interleukin-3, or thrombopoietin (Tpo). Independent of the cytokine, the cells constitutively expressed a well-defined megakaryocyte phenotype, with 70-95% of the cells positive for CD4, CD34, and platelet glycoproteins Ib, IIb, and IIIa. Fewer than 10% of the cells had detectable erythroid glycophorin A. Erythropoiesis was induced in HU-3 parental cells and five clones harvested from culture medium containing GM-CSF by replacement of the growth-promoting cytokine with stem cell factor (SCF) and erythropoietin (Epo). During the first week of induction, the proliferating cells slowly acquired erythroid markers. Concomitant with a maturational growth arrest during the second week, there was a rapid accumulation of gamma and beta globin chains and benzidine reactive hemoglobin, as well as a distinct erythroid morphology. The culture declined after 12 days because of the transient effect of SCF in maintaining viability. Parental and cloned cells cultured for 7 days in Tpo-supplemented medium responded to the synergistic growth effect of SCF and Epo but were markedly suppressed in their yield of hemoglobinized cells. Recycling of the cells in GM-CSF for 4 days did not reverse the suppressive effect of Tpo. These results suggest a role for Tpo in the lineage commitment of erythromegakaryocytic progenitors by suppressing the erythroid potential. With its constitutive megakaryocyte phenotype and inducible erythroid potential, the self-renewing bipotential HU-3 cell line may represent one of the earliest stages in megakaryocytopoiesis before irreversible lineage commitment. The suppressive effect of Tpo on the erythroid potential of cloned HU-3 cells enhances the value of this cell line for deciphering the molecular and cellular events during lineage commitment of progenitor cells.

Cytokine mRNA expression in human platelets and a megakaryocytic cell line and cytokine modulation of platelet function.

Platelet formation and function are regulated, in vivo, to varying degrees by cytokines in the micro-environment. While white blood cells are the major source of cytokines within the cardiovascular system, the question addressed in this study was whether platelets and the platelet precursor, the megakaryocyte, may also serve as a source of cytokines. Cytokines produced by or carried within platelets could be released at sites of vascular injury and participate in wound healing. Platelets and a human megakaryocyte-like cell line, HU3, were found to express message for interleukin 7 (IL-7), stem cell factor (SCF), transforming growth factor beta (TGF-beta), cMpl, the IgE receptor subunits Fc epsilon RI alpha gamma and the transcription factor, NF-E2. Other cytokines expressed in HU3 cells but not in platelets included IL-1 beta, IL-6, IL-10, IL-13, TNF-alpha and the FC epsilon RI beta subunit. The HU3 cell line seemed to be further along the maturation/differentiation pathway to platelet formation than a second blood derived bipotential cell line, MB02. The MB02 cell line did not express IL-6, IL-10, SCF, TNF-omega nor cMpl. Furthermore, culturing the HU3 cells in TPO appeared to repress expression of Fc epsilon RI beta directing the cell closer to the platelet phenotype. In light of the presence of cytokine expression in platelets/megakaryocytes, agonist-induced platelet aggregation was measured in the presence of added cytokines as a means to evaluate potential cytokine modulation of platelet function. Collagen-induced aggregations were significantly enhanced by IL-6, SCF and TPO. Other cytokines tested significantly stimulated the thrombin receptor activating peptide, SFLLRNP-, U46619- and ADP-induced platelet aggregations with TPO being the most consistent activator. It is possible that cytokines released from platelets act in concert with cytokines released from other cellular sources to modulate haemostasis and thrombosis differentially depending upon the site of injury.

ATP modification of serotonin-induced contraction of the rat pulmonary artery.

Serotonin (5HT) and ATP are simultaneously released from activated platelets at the site of vascular injury and are hypothesized to play a significant role in hemostasis. Our laboratory investigated the modulation of vascular contraction of arterial ring segments by 5HT plus ATP as a model of the potential regulation of localized vascular tone by platelet releasates in regions of arterial damage. This study expands our focus on how these two vasoactive components, released from platelet dense granules, regulate vascular tone. 5HT- and 5HT analog-induced vasoconstrictions were measured in the presence or absence of ATP and ATP analogs with intact or deendothelialized rat pulmonary arterial ring segments suspended in organ baths. The possible presence of 5HT2 and 5HT1A receptor types in the rat pulmonary artery was demonstrated by vasoconstrictions induced by 5HT and (+)-8-hydroxy-2-(di-N-propylamino) tetralin hydrobromide (DPAT). The DPAT response was only 30%-50% of that induced by comparable concentrations of 5HT. The 5HT-induced contraction was inhibited by the 5HT2 antagonist, ketanserin. ATP equally relaxed 5HT and DPAT contracted tissue while the P2X agonist, alphabeta-methylene ATP, increased the contracted state of DPAT-treated arteries to a significantly greater extent than observed with 5HT. The P2y agonist, 3'-O-(4-benzoyl)benzoyl ATP (BzATP), the P2X agonist betagamma-methylene ATP, and ATP all relaxed 5HT-induced contractions to similar levels under a number of physiological conditions. The final level of 5HT-induced tissue contraction was the same whether ATP was added prior to, after, or simultaneously with 5HT. ATP and the phosphodiesterase inhibitor, theophylline, inhibited 5HT-induced vasoconstriction in an additive fashion. The ATP effects were endothelium dependent, while the inhibition by theophylline was not. The distribution of 5HT and ATP receptor types, as indicated by these and numerous other studies, appears to vary within different regions of the cardiovascular system. Extracellular ATP can synergistically enhance or inhibit 5HT-contracted blood vessels differentially at localized regions, which would significantly impact on localized vascular tone, and this in turn may modulate hemostasis and thrombosis.

A possible dual physiological role of extracellular ATP in the modulation of platelet aggregation.

ATP and ADP are simultaneously released from activated platelets in equimolar concentrations. Micromolar concentrations of ATP inhibit platelet aggregation by both competitive and non-competitive mechanisms. The current studies addressed the question of how platelets respond to agonists in the presence of nanomolar and micromolar concentrations of ATP and ADP alone or in combination. This is a significant issue since the concentration of ATP +/- ADP may vary widely within a microenvironment depending upon the source and cause for the release of the nucleotides. ATP (1-10 nM) was found to significantly enhance the thromboxane A2 analog, U44619-, collagen- and thrombin-induced platelet aggregations. Conversely, ATP at 1-100 microM inhibited these same reactions. ADP, in general, behaved exactly opposite to ATP. When equal amounts of ATP and ADP were added together the ADP response appeared to predominate. The observed ATP-induced response was not due to a hydrolytic product as evidenced by an unaltered response to ATP in the presence of adenosine deaminase or the ATP generating system, creatine phosphate plus creatine phosphokinase. Adenosine (1-10 nM), like ADP, inhibited agonist-induced platelet aggregation. The stimulation of agonist-induced platelet aggregation by 1-10 nM extracellular ATP appears to depend upon the phosphorylation of platelet membrane ecto proteins. The ATP analog, beta gamma-methylene ATP, that is incapable of serving as a phosphate donor for protein kinases, inhibited rather than stimulated agonist-induced platelet aggregation. The dual response of platelets to low and high concentrations of extracellular ATP +/- ADP may play a physiological role in hemostasis and thrombosis under normal and pathological conditions.

Characterization of a potent platelet aggregation inducer from Cerastes cerastes (Egyptian sand viper) venom.

A potent, proteinaceous inducer of platelet aggregation designated as IVa, has been purified to homogeneity from Cerastes cerastes venom by molecular sieve and ion exchange chromatography. It is composed of 2 subunits with total M(r) of 62,000 as shown by native gel chromatography and chemical cross-linking with disuccinimidyl suberate. It is not clear at the present time whether both subunits are identical gene products, however, both have identical N-terminal sequences for the first 15 amino acids. The protein has a pI above 9.6. IVa (0.1 micrograms/ml) could aggregate platelets up to 80% and was inhibited by p-APMSF, leupeptin, iodoacetamide, protein kinase C inhibitor, phosphatase inhibitor, ATP and PGE1, while it was insensitive to acetylsalicylic acid, ADP scavenger system, protein kinase A inhibitor and hirudin. Protein IVa is a serine proteinase with thrombin-like activity as it hydrolysed thrombin chromogenic substrate CBS 34.47, its aggregatory activity was partially inhibited by monoclonal antibodies against GPIb and the thrombin receptor, as was the thrombin, and its ability to induce intracellular Ca2+ release was blocked by pretreating platelets with thrombin. Unlike thrombin, the IVa protein showed very weak coagulant activity as indicated by plasma recalcification time and fibrinogen clotting time although it could hydrolyse fibrinogen alpha-chains.

Extracellular ATP inhibits agonist-induced mobilization of internal calcium in human platelets.

Our previous studies have demonstrated that platelets possess ATP purinergic receptors in addition to the ADP, P2T, receptor. Occupancy of the P2 receptor by ATP inhibited agonist-induced platelet aggregation. This study demonstrated that the mechanism of inhibition may involve ATP inhibition of agonist-induced mobilization of internal calcium. Within the cardiovascular system, the ATP inhibition of calcium mobilization is unique to platelets. All other cell types in the cardiovascular system, where calcium mobilization is affected by extracellular ATP, responded with an increased mobilization as opposed to inhibition. The platelet inhibitory response to ATP was enhanced by the addition of an ATP generating system, creatine phosphate/phosphocreatine kinase. ATP and ATP analogues were found to inhibit calcium mobilization with a rank order of alpha beta-methylene ATP, beta gamma-methylene ATP approximately ATP > benzoyl ATP > 2 methylthio ATP which is a characteristic of P2x-like receptors. The inhibitory effect of ATP could be abrogated by prolonged treatment of platelets with the P2x desensitizing agent, alpha beta-methylene ATP. Also, UTP and CTP were approximately as effective inhibitors as ATP while GTP was not. ATP competition with ADP for the P2T receptor was excluded in studies with platelets derived from an aspirin-treated individual which were essentially insensitive to ADP. The agonist-induced calcium mobilization and inhibition by ATP occurred with the thromboxane A2 mimetic, U46619, collagen and thrombin; however, the kinetics of mobilization varied somewhat with the different agonists. The responses to extracellular ATP were independent of extracellular Ca2+, where 1 mM calcium or 0.3 mM EGTA was added to the reaction mixture. The inhibition of calcium mobilization coupled to inhibition of platelet aggregation by extracellular ATP may serve an important physiologic role. ATP, released from activated platelets at localized sites of vascular injury, may help to limit the size of the platelet plug-clot that, if left unregulated, could occlude the injured blood vessel.

Aggregation of human and canine platelets: modulation by purine nucleotides.

This study compared the responses of canine and human platelets to various aggregating agonists in the presence or absence of extracellular ATP and ATP analogues. Canine and human platelets were approximately equally reactive with ADP or collagen while the canine platelets were about 10 fold more sensitive to thrombin. Canine platelets were insensitive to the thromboxane mimetic U46619 but were synergistically aggregated by a mixture of ADP and U46619. Human platelets were very sensitive to U46619. Aggregations of human platelets with all of the above agonists were inhibited by extracellular ATP; beta, gamma methylene ATP (beta gamma ATP) and benzoyl ATP (BzATP) with a rank order suggestive of an interaction with P2x-like purinoceptors which support our previous findings. The comparable aggregations of canine platelets were likewise inhibited by ATP and its analogues but with a rank order suggestive of an interaction with P2y-like purinoceptors. ATP inhibited U46619- and ADP-induced aggregation of human platelets and ADP-induced aggregation of canine platelets, presumably, in part, due to competition for the ADP P2T receptor. However, when U46619 was added to either ATP or ATP analogue-inhibited ADP-treated canine platelets, the inhibition was nullified. Furthermore, we demonstrated, for the first time, that the canine thromboxane receptor becomes reactive to U46619 alone after incubation at room temperature for 3.5-5 hrs while human platelets become inactive under similar conditions. The implication of these studies is that there are significant differences in the canine and human platelet thromboxane and purine receptors. The future characterization of these differences and the mechanism by which they function should further our understanding of the impact of extracellular ATP on hemostasis and thrombosis.

Immunological identification of human platelet mitochondrial DNA topoisomerase I.

A nuclear human blood platelets have been used to study mitochondrial topoisomerase activity in the absence of nuclear contamination. Previous work utilizing this novel system demonstrated that platelet mitochondria contain type-I topoisomerase (Kosovsky, M.J. and Soslau, G. (1991) Biochim. Biophys. Acta 1078, 56-62). The present work has demonstrated that mitochondrial topoisomerase activity was inhibited by the specific topoisomerase-I inhibitor, topotecan, yet was not affected by a specific topoisomerase-II inhibitor, VM-26. These results confirm that platelet mitochondria contain topoisomerase I, yet do not contain a detectable level of topoisomerase-II activity. It has been demonstrated for the first time that antibodies directed against nuclear topo I cross-react with mitochondria topo I. Furthermore, immunoblot analysis of platelet mitochondrial proteins, in conjunction with renaturation studies, has led to the identification of a catalytically active 60-kDa form of mitochondrial topoisomerase I.

Occupancy of P2 purinoceptors with unique properties modulates the function of human platelets.

This report demonstrates that platelets possess P2 purinoceptors with unique properties that distinguish them from the ADP (P2T) receptor. Extracellular ATP, and its poorly hydrolyzable analogues, inhibit collagen- and U46619 (a thromboxane mimetic)-induced platelet aggregations. Adenosine deaminase was without effect on ATP action while reversing the inhibitory effect of adenosine. A unique aspect of the P2 receptor is the sensitivity to UTP and CTP and insensitivity to GTP. The rank order of inhibition by beta gamma-methylene ATP, alpha beta-methylene ATP > ATP indicates that a P2x-like receptor is present on the platelet membrane. This conclusion is further supported by the nearly complete desensitization to ATP by pre-exposure of platelets to alpha beta-methylene-ATP. However, unlike previously described P2x purinoceptors, the inhibition of platelet aggregation by extracellular ATP appears to result, at least in part, from the ATP-induced increase of intracellular cyclic AMP levels apparently coupled through a Gs protein. The combined addition of iloprost (0.14 to 1.39 nM) and ATP (18 microM) or ATP (20-40 microM) and the phosphodiesterase inhibitor theophylline (0.5 to 1 mM) synergistically inhibited platelet aggregation implying a common interactive site with adenylate cyclase. This is further substantiated by the ability of the adenylate cyclase inhibitor, 2',5'-dideoxyadenosine, to abrogate the inhibitory effects of ATP. The protein kinase A (PKA) inhibitor H1004 blocks ATP inhibition of platelet aggregation while the protein kinase C inhibitor H7 did not. This implies that the generation of cyclic AMP, with the subsequent activation of PKA and phosphorylation of selected proteins is required, in part, for the action of ATP.

Characterization of the phosphorylated state of protein 4.2 from a patient partially deficient in protein 4.2.

These studies compare the protein 4.2 found in a patient with osmotically fragile, spherocytic erythrocytes to the normal protein 4.2. The patient protein 4.2 is present in the erythrocyte ghost membranes as a doublet of 74 and 72 KDa at a concentration less than 1% of normal. The patient protein 4.2 becomes highly phosphorylated in the presence of Zn++ and is phosphorylated, relative to the amount of protein present, to a greater extent than the normal 72 KDa protein 4.2. These studies indicate that both the patient and the normal protein 4.2 usually exists in a highly phosphorylated state. The phosphorylation sites on the patient protein 4.2 appear to be more readily cycled than on the normal protein 4.2. Staphylococcus aureus V8 protease generates similar phosphopeptides in both the normal and patient protein 4.2 except for an extra 11 KDa phosphopeptide generated from the 74 KDa form of the protein.

Zinc ions and alkaline pH alter the phosphorylated state of proteins 3 and 4.2 in human erythrocyte membranes.

The phosphorylation patterns of isolated red blood cell (RBC) membranes labeled with [gamma-32P] ATP are altered by Zn++ ions. Zn++ ions caused an increased phosphate incorporation into a 72 KDa protein and several proteins in the 40-60 KDa region and a decrease in the labeling of a 53 KDa protein. The 72 KDa and 53 KDa proteins have been identified as protein 4.2 and a protease-cleaved fragment of protein 3, respectively. Evidence suggests that the changes in phosphorylation pattern may be due to the stimulation of endogenous membrane alkaline phosphatase(s). Our results suggest that Zn++, at physiological concentrations in the intact erythrocyte, could modulate the phosphorylation of selected proteins which may regulate their association in the cytoskeletal network.

The bioluminescent detection of platelet released ATP: collagen-induced release and potential errors.

The bioluminescent detection of ATP released from activated platelets is an important diagnostic and experimental assay. Potential errors in the interpretation of the data may be introduced due to the lability of luciferin-luciferase and the amount of platelet agonist employed. Loss of luciferin-luciferase activity is temperature dependent with a 50% decrease in activity in 1-4 min at 37 degrees C. Plasma components do not appear to contribute to the inactivation of the detection system. Due to the significant loss of enzyme activity at variable times, the method of standardizing ATP concentrations is crucial for the accurate determination of ATP released from activated platelets. A nearly 5-fold error is introduced into the routinely employed assay procedure where the standard ATP concentration is determined 5 min after the addition of agonist. This report demonstrates that the standard ATP concentration must be determined with a separate platelet sample at the same time as the ATP was released from the agonist-induced experimental platelet sample. A second significant error in the assay system may be introduced by the agonist concentration employed even when the final level of aggregation is the same. When collagen is employed as the agonist the amount of ATP released appears to depend, in part, on the initial intensity of the aggregation response and not on collagen type (Type I versus IV). The corrective procedures described here for the detection of ATP are not likely to change the qualitative results of most studies but would significantly alter the quantitative results.

Effect of tranexamic acid on platelet ADP during extracorporeal circulation.

Seventeen adults received the antifibrinolytic drug tranexamic acid during cardiac surgery utilizing extracorporeal circulation (ECC). In 8 patients, drug administration began prior to skin incision (pre-ECC); infusions commenced after ECC and protamine administration in another 9 patients (post-ECC). Compared with the post-ECC group, the pre-ECC group exhibited less bleeding via mediastinal drains (420 vs. 655 mL/12 h median, P = 0.024), decreased frequency of the presence (greater than or equal to 10 micrograms/mL) of fibrin split products (P less than 0.05), and greater platelet dense granule content of adenosine diphosphate after surgery (15.47 vs. 4.05 nmoles/mg protein median, P = 0.021). Follow-up in vitro study of tranexamic acid inhibition of plasmin-induced platelet activation utilizing normal human platelet rich plasma and porcine plasmin revealed a 13-fold lower concentration of tranexamic acid for 50% inhibition when plasmin was preincubated with the drug (1.2 micrograms/mL, 95% CI = 1.13-1.60 micrograms/mL) compared to when platelet rich plasma was preincubated with the drug (16 micrograms/mL, 95% CI = 7.3-99. micrograms/mL). Plasmin inactivated with tranexamic acid retained its ability to inhibit thrombin-induced platelet activation, thus suggesting that tranexamic acid inhibits plasmin's catalytic activity and not its binding to platelets. Both clot lysis and platelet dysfunction may contribute to bleeding after ECC. Tranexamic acid blocks plasmin-induced partial platelet activation during ECC, thus preserving platelet function and promoting hemostasis after ECC.

Mitochondrial DNA topoisomerase I from human platelets.

An anucleated cell system has been used for the first time to study mitochondrial topoisomerase activity. Mitochondrial extracts from human blood platelets contained type I topoisomerase. The type I classification was based on ATP-independent activity, inhibition by ATP or camptothecin, and the lack of inhibition by novobiocin. Platelet mitochondrial topoisomerase I relaxation activity was inhibited linearly by increasing concentrations of EGTA. Topoisomerase activity greater than 90% inhibited by 175 microM EGTA was partially restored to 16 and 50% of the initial level of activity by the subsequent addition of 50 and 100 microM Ca2+, respectively. Additionally, results from studies of partially purified platelet mitochondrial topoisomerase I were consistent with the crude extract data. This work supports the hypothesis that platelet mitochondria contain a type I topoisomerase that is biochemically distinct from that previously isolated and characterized from cell nuclei.