Biomedical subjects

S Niewiarowski

Publications and source records attributed to S Niewiarowski.

At least 37 records · Page 2Linked to original sources

PubMed1998

Alboaggregins A and B. Structure and interaction with human platelets.

Viper venoms contain a variety of platelet binding proteins including those which bind to platelet GPIb/GPIX. Most of these proteins inhibit von Willebrand factor mediated platelet agglutination. Here we report the primary structures of unique members of this family, alboaggregins A and B, isolated from Trimeresurus albolabris, which have the ability to stimulate platelet agglutination and aggregation. Four chains of alboaggregin A and two chains of alboaggregin B share a high degree of homology and all cysteines in both alboaggregins are conserved. Both alboaggregins caused similar agglutination of fixed platelets. Alboaggregin A induced platelet aggregation and release reaction with EC50 = 10 and 30 nM, respectively, which is 20-fold lower than those for alboaggregin B. These observations suggest that the dimeric structure of alboaggregin B is sufficient to mediate its binding to GPIb and induce agglutination of platelets whereas aggregation and release reaction are significantly enhanced by tetrameric structure of alboaggregin A.

Amino Acid Sequence↗

PubMed1998

Measurement of platelet microparticles during cardiopulmonary bypass by means of captured ELISA for GPIIb/IIIa.

A captured enzyme-linked immunosorbent assay (ELISA) using the disintegrin, kistrin, is described and used to measure platelet microparticles (PMP) generated during open heart surgery. This ELISA detects 75 ng/ml of glycoprotein IIb/IIIa (GPIIa/IIIa) in solution and is more sensitive and less variable than flow cytometry and radioimmunoassay. By ELISA, mean values of GPIIb/IIIa in PMP are 14.2 +/- 7.9 microg/ml for outdated platelets and 0.28 +/- 0.1 microg/ml in fresh blood from healthy donors. Normal washed platelets (10(8)) contain 8.8 microg of GPIIb/IIIa. In 12 cardiac surgical patients, PMP measured by ELISA significantly increased (p = 0.039) to 0.58 +/- 0.3 microg/ml at the end of cardiopulmonary bypass, but the increase measured by flow cytometry (1207 to 1447 events in PMP gate) was not significant. Neither heparin nor protamine alter PMP. After cardiopulmonary bypass, PMP concentrations return to baseline values before protamine is given. Concentrations of PMP in pericardial blood are greater than in simultaneous perfusate. This ELISA is more sensitive and accurate than alternate methods for measuring PMP and shows the PMP production and rapid clearance during open cardiac surgery.

Cardiopulmonary Bypass↗

PubMed1998

Thrombin and human plasma kallikrein inhibition during simulated extracorporeal circulation block platelet and neutrophil activation.

Cardiopulmonary bypass causes hemorrhagic complications, and initiates a chemical and cellular inflammatory response. Contact of blood with synthetic surfaces leads to qualitative and quantitative alterations in platelets, neutrophils, complement, and contact systems. Despite the fact that cardiopulmonary bypass is carried out in the presence of high doses of heparin, there is significant activation of both platelets and neutrophils. Thrombin is protected on cell and fibrin surfaces from antithrombin, even in the presence of high doses of heparin (approximately 5 U/ml). We therefore studied the effect of a small (Mr = 497), highly effective (Ki = 41 pM), reversible tripeptide inhibitor of thrombin, DUP 714 (1 microM), in a well characterized model of simulated extracorporeal circulation. In the absence of DUP 714, platelet counts decreased by 75% 5 min after the start of extracorporeal bypass and increased to 48% at 120 min of recirculation. DUP 714 significantly preserved platelet counts, decreased plasma levels of platelet beta-thromboglobulin levels, but did not prevent a decrease in sensitivity of platelets to adenosine diphosphate. Kallikrein-C1-inhibitor and C1-C1-inhibitor complexes increased progressively from 0.32 U/ml to 0.67 U/ml and from 4.45 U/ml to 7.25 U/ml, respectively, during 120 min of recirculation without DUP 714. Addition of DUP 714 significantly inhibited kallikrein-C1-inhibitor complex formation but did not affect C1-C1-inhibitor complexes. In the absence of DUP 714, human neutrophil elastase levels rose from a baseline of 0.01 +/- 0.00 microg/ml to 1.18 +/- 0.21 microg/ml during 120 min of recirculation. Human neutrophil elastase release at 120 min was significantly inhibited in the presence of DUP 714 to 37% of the value with heparin alone. These results indicated that addition of this novel thrombin (and kallikrein) inhibitor to heparin preserved platelet counts, decreased platelet secretion, and provided the additional benefit of partially blocking neutrophil activation during simulated extracorporeal circulation.

Cardiopulmonary Bypass↗

PubMed1998

Interaction of leukocytes with platelet microparticles derived from outdated platelet concentrates.

Platelet microparticles (PMP) were isolated from outdated platelets by a combination of differential centrifugation and gel filtration, and the concentration of PMP was expressed in the equivalent of GPIIb/IIIa complex measured by captured ELISA. PMP bound to isolated neutrophils and macrophages in a dose-dependent manner, but they did not bind to lymphocytes. Incubation of PMP with neutrophils did not activate these cells as measured by up-regulation of Mac-1, release of human granulocyte elastase, and calcium mobilization. Incubation of PMP with macrophages did not enhance IL-8 production and the oxygen burst but slightly and significantly increased production of MCP-1. After 10 min incubation of PMP with macrophages, an increase of GPIIb/IIIa antigen was observed suggesting that PMP may be endocytosed by macrophages. In conclusion, PMP bind to leukocytes, but, in contrast to activated platelets, do not play a significant role in leukocyte activation.

Biomarkers↗

PubMed1997

The disulphide bond pattern of bitistatin, a disintegrin isolated from the venom of the viper Bitis arietans.

The disulphide bond pattern of the long disintegrin bitistatin (83 amino acids, 14 cysteines) was established using structural information gathered by amino acid analysis, N-terminal sequencing, and molecular mass determination of fragments isolated by reversed-phase HPLC after polypeptide degradation with trypsin and oxalic acid. A computer program was used to calculate all possible combinations of disulphide-bonded peptides matching the mass spectrometric data, and the output was filtered using compositional and sequence data. Disulphide bonds between cysteines 16-34, 18-29, 28-51, 42-48, 47-72, and 60-79 are conserved in medium-long disintegrins flavoridin and kistrin (70 amino acids, 12 cysteines), and the two cysteine residues at positions 5 and 24 found in bitistatin but not in other disintegrin molecules are disulphide-bridged. This linkage creates an extra, large loop, which, depending on whether the NMR structure of flavoridin or kistrin is used for modelling the structure of bitistatin, lies opposite or nearly parallel, respectively, to the biologically active RGD-containing loop.

Amino Acid Sequence↗

PubMed1997

Significance of RGD loop and C-terminal domain of echistatin for recognition of alphaIIb beta3 and alpha(v) beta3 integrins and expression of ligand-induced binding site.

Echistatin is a viper venom disintegrin containing RGD loop maintained by disulfide bridges. It binds with a high affinity to alpha(v) beta3 and alphaIIb beta3 and it induces extensive conformational changes in these integrins resulting in expression of ligand-induced binding site (LIBS) epitopes. We investigated the activities of echistatin and its three analogues (R24A, D27W, echistatin 1-41). R24A echistatin did not react with alphaIIb beta3 and alpha(v) beta3 integrins and did not cause LIBS effect. D27W echistatin showed increased binding to alphaIIb beta3 and decreased binding to alpha(v) beta3. This substitution impaired the ability of echistatin to induce LIBS in alpha(v) beta3 integrin. Deletion of nine C-terminal amino acids of echistatin decreased its ability to bind alphaIIb beta3 and inhibit platelet aggregation. Truncated echistatin failed to induce LIBS epitopes on cells transfected with alphaIIb beta3 and alpha(v) beta3 genes. The ability of echistatin 1-41 to compete with binding of vitronectin to immobilized alpha(v) beta3 and monoclonal antibody 7E3 to platelets and to VNRC3 cells was decreased, although this analogue, after immobilization, retained its ability to bind purified alpha(v) beta3. We propose a hypothesis in which echistatin's RGD loop determines selective recognition of alphaIIb beta3 and alpha(v) beta3 integrin, whereas the C-terminal domain supports its binding to resting integrin and significantly contributes to the expression of LIBS epitope and to conformational changes of the receptor, leading to a further increase of the binding affinity of echistatin and of the inhibitory effect.

Animals↗

PubMed1997

A natural motif approach to protein design: a synthetic leucine zipper peptide mimics the biological function of the platelet factor 4 protein.

The design of smaller functional mimics of large proteins has long been an important challenge. In this study we use the natural leucine zipper as a structural template to design a 31-residue peptide analog that mimics the function of the larger platelet factor 4 (PF4) protein. The heparin binding activity of PF4 has been introduced into an unrelated leucine zipper sequence only by virtue of incorporating four lysines of PF4. Circular dichroism and binding experiments have shown that the designed leucine zipper peptide adopts a stable helical conformation and shows significant PF4-like heparin binding activity. These results strongly suggest that the lysine residues play an important role in the binding of PF4 to heparin. The de novo generation of the PF4 function in a designed leucine zipper peptide demonstrates that the leucine zipper motif is a useful scaffold for the design of functional peptides and proteins.

Amino Acid Sequence↗

PubMed1997

Interaction of beta 3 integrin-derived peptides 214-218 and 217-231 with alpha IIb beta 3 complex and with fibrinogen A alpha-chain.

beta 3 integrin-derived peptides 214-218 and 217-231 have been shown previously to inhibit platelet aggregation and fibrinogen binding to platelets and to purified receptor. In this paper we study the activity of both peptides in inhibition of binding of biotinylated fibrinogen to activated platelets and to immobilized alpha IIb beta 3 receptor. We found that the mechanism of this inhibition by both peptides is different 125I-labeled 214-218 peptide binds to alpha IIb beta 3 but in contrast, 125I-labeled 217-231 peptide binds to the A alpha-chain of native and gamma' fibrinogen, as judged by the cross-linking study. In solid phase assay both purified alpha IIb beta 3 and 217-231 peptide bound extensively to native and recombinant fibrinogen, and to fibrinogen with either D574E or D97E mutations in the A alpha-chain. Binding of purified alpha IIb beta 3 to gamma' fibrinogen was markedly impaired whereas binding of 217-231 was only slightly impaired in comparison with native fibrinogen. Binding of 217-231 to fibrinogen fragment X was also reduced suggesting that sequences other than RGDS and RGDF may represent binding sites for this peptide. We hypothesize that the close vicinity of fibrinogen binding site (217-231) and of the site participating in conformational changes of the alpha IIb beta 3 receptor (214-218) may facilitate fibrinogen interaction with its receptor.

Adenosine Diphosphate↗

PubMed1997

A baboon model for hematologic studies of cardiopulmonary bypass.

Objective investigation of new inhibitors of blood protein or cellular systems that are activated during cardiopulmonary bypass (CPB) is impeded by the absence of a satisfactory animal model. Because most baboon hematologic proteins immunologically cross-react with those used for human assays, we developed a robust, reusable baboon model of CPB. Blood samples were obtained from adult baboons at six time intervals before, during, and after 60 minutes of partial CPB at 37 degrees C with peripheral cannulas. Both membrane (n = 7) and bubble oxygenators (n = 7) were investigated. We measured platelet and white blood cell counts; platelet response to adenosine diphosphate and release of beta-thromboglobulin; fibrinopeptide A, prothrombin fragment F1.2, thrombin-antithrombin complex, D-dimer, and plasmin-antiplasmin complex; activated complement (C3b/c and C4b/c); elastase-alpha1 proteinase inhibitor complex; and bleeding times. Adherent glycoprotein IIIa antigen in Triton X-100 washes of the perfusion circuit was also measured. Markers of baboon platelet, complement, and neutrophil activation and thrombosis significantly increased during CPB with bubble oxygenator systems but did not change appreciably in membrane oxygenator circuits. Markers of fibrinolysis, D-dimer, and plasmin-antiplasmin complex did not change with either oxygenator. The baboon model of CPB, when a bubble oxygenator is used, is a robust, reusable animal model for evaluating inhibitors of platelet, complement, and neutrophil activation and thrombosis during and after CPB.

Adenosine Diphosphate↗

PubMed1997

Tirofiban provides "platelet anesthesia" during cardiopulmonary bypass in baboons.

OBJECTIVE: Tirofiban (Aggrastat) is a reversible, nonpeptide inhibitor of platelet glycoprotein II/IIIa receptors. We tested the hypothesis that tirofiban preserves platelet number and function and shortens postoperative bleeding times in baboons after cardiopulmonary bypass. METHODS: Four groups were studied: control, n = 12; low-dose tirofiban (0.1 microg/kg per minute), n = 7; high-dose tirofiban (0.3 microg/kg per minute), n = 7; and bolus tirofiban (15 microg/kg) followed by 0.1 microg/kg per minute during cardiopulmonary bypass, n = 7. After heparin, animals were perfused for 60 minutes at 50 ml/kg per minute and 37 degrees C with a bubble oxygenator, roller pump, and peripheral cannulation. Hemodynamics, platelet count, platelet aggregation to adenosine diphosphate, and release of beta-thromboglobulin were measured before tirofiban infusion, before heparin, after heparin before bypass, after 5 and 55 minutes of bypass, after protamine, and 60 minutes after protamine. Template bleeding times were measured at the same times except during cardiopulmonary bypass and 120 and 180 minutes after protamine administration. Platelet glycoprotein IIIa antigen was measured in Triton X-100 washes (Sigma Chemical Company) of the perfusion circuit after bypass. RESULTS: High-dose tirofiban completely prevents platelet loss during cardiopulmonary bypass. beta-Thromboglobulin release and sensitivity to adenosine diphosphate are significantly less than control at the end of bypass in all tirofiban groups. Template bleeding times return to preoperative values in both the low- and high-dose tirofiban groups 180 minutes after protamine administration and are significantly less than control bleeding times at both 120 and 180 minutes after protamine. Surface glycoprotein IIIa antigen does not significantly differ between groups. CONCLUSION: High-dose tirofiban completely preserves platelet number and improves platelet function during cardiopulmonary bypass in baboons and significantly accelerates restoration of normal template bleeding times after bypass.

Animals↗

PubMed1996

Effect of factor Xa inhibitors on thrombin formation and complement and neutrophil activation during in vitro extracorporeal circulation.

BACKGROUND: Even when large doses of heparin are administered during cardiopulmonary bypass, thrombin is produced. Thrombin is a powerful protease that is associated with the thrombotic and bleeding complications of open heart surgery and is produced by cleavage of prothrombin by factor Xa. This study assessed the ability of a specific inhibitor of factor Xa, recombinant tick anticoagulant peptide (rTAP), alone or in combination with standard heparin and a low-molecular-weight heparin, enoxaparin, to suppress thrombin formation and activity during in vitro extracorporeal circulation. METHODS AND RESULTS: Fresh, anticoagulated human blood was recirculated for 2 hours in an extracorporeal membrane oxygenator perfusion circuit at 37 degrees C. Four anticoagulant protocols were evaluated; porcine heparin (3.75 U/mL); enoxaparin (17.5 U/mL); rTAP (4 mumol/L); and porcine heparin plus rTAP (2 mumol/L). Blood samples were obtained for analysis from the donor, after anticoagulation, and after 5, 30, 60, and 120 minutes of recirculation. There were no significant differences between groups in platelet count, response to adenosine diphosphate, or prothrombin fragment (F1.2) production. rTAP plus heparin reduced beta-thromboglobulin release; fibrinopeptide A concentrations were significantly higher with rTAP alone. Enoxaparin strongly and significantly inhibited complement C5b9 production and neutrophil elastase release and was associated with significantly increased concentrations of C1-C1 inhibitor and kallikrein-C1 inhibitor complexes. CONCLUSIONS: rTAP does not reduce thrombin formation or activity during in vitro extracorporeal circulation. Enoxaparin markedly inhibits formation of the complement membrane attack complex and neutrophil elastase release, possibly by accelerating C1 inhibitor activity.

Adenosine Diphosphate↗

PubMed1996

Importance of the structure of the RGD-containing loop in the disintegrins echistatin and eristostatin for recognition of alpha IIb beta 3 and alpha v beta 3 integrins.

Echistatin and eristostatin are structurally homologous distintegrins which exhibit significant functional differences in interaction with various integrins. We hypothesized that this may reflect differences in the sequences of their RGD loops: 20CKRARGDDMDDYC32 AND 23CRVARGDWNDDYC35, respectively. Mapping of eristostatin peptides obtained by proteolytic digestion suggested that it has the same alignment of S-S bridges as echistatin. Synthetic echistatin D27W resembled eristostatin since it had increased platelet aggregation inhibitory activity, increased potency to block fibrinogen binding to alpha IIb beta 3, and decreased potency to block vitronectin binding to alpha v beta 3 as compared to wild-type echistatin. Since eristostatin and echistatin have a similar pattern of disulfide bridges, we constructed molecular models of eristostatin based on echistatin NMR coordinates. The RGD loops of eristostatin and echistatin D27W were wider than echistatin's due to the placement of tryptophan (rather than aspartic acid) immediately after the RGD sequence. We propose a hypothesis that the width and shape of the RGD loop are important ligand structural features that affect fitting of ligand to the binding pocket of alpha IIb beta 3 and alpha v beta 3.

Amino Acid Sequence↗

PubMed1996

Immunological characterization of eristostatin and echistatin binding sites on alpha IIb beta 3 and alpha V beta 3 integrins.

Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with alpha IIb beta 3 genes (A5 cells) and to CHO cells transfected with alpha v beta 3 genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (Kd 28 nM) and to 200000 sites (Kd 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (Kd 53 nM) per A5 cell and to 443000 sites (Kd 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the beta 3 subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (alpha IIb beta 3 complex dependent) and 7E3 (alpha IIb beta 3 and alpha v beta 3 complex dependent) to A5 cells, to resting and to activated platelets and to purified alpha IIb beta 3. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on alpha IIb beta 3 integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on alpha v beta 3 integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified alpha v beta 3 suggesting that alpha v beta 3 and alpha IIb beta 3 might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on alpha v beta 3.

Animals↗

PubMed1996

Disintegrin interaction with alpha V beta 3 integrin on human umbilical vein endothelial cells: expression of ligand-induced binding site on beta 3 subunit.

The effect of seven disintegrins (albolabrin, barbourin, bitistatin, echistatin, eristostatin, flavoridin, and kistrin) and the neurotoxin analogue, mambin, on the adhesion of human umbilical vein endothelial cells (HUVEC) to immobilized vitronectin and fibronectin has been studied. Adhesion to vitronectin was significantly inhibited by echistatin, kistrin, flavoridin, and mambin. Echistatin, flavoridin, and kistrin bound with high affinity to immobilized alpha V beta 3 in solid phase assay; other disintegrins bound at a much lower level. Echistatin and flavoridin had a modest inhibitory effect on HUVEC adhesion to fibronectin. HUVEC adhered to disintegrins with a high selectivity toward bitistatin, echistatin, flavoridin, kistrin, and mambin. Adhesion of HUVEC to fibronectin and vitronectin resulted in cell spreading, whereas cells adhering to immobilized echistatin remained globular and cells adhering to kistrin showed abnormal morphology. Echistatin and kistrin potently inhibited the binding of monoclonal antibody (Mab) 7E3, which recognizes the alpha V beta 3 complex, to HUVEC. Echistatin and kistrin also induced the binding to HUVEC of Mab 62, which recognizes the ligand-induced binding site (LIBS) epitope on the beta 3 subunit, enhancing HUVEC binding to immobilized Mab 62. Similar results with both antibodies were obtained in Chinese hamster ovary cells transfected with alpha V beta 3 genes. In conclusion, disintegrin interaction with HUVEC appears to be selectively mediated by alpha V beta 3 receptors, and it results in an expression of LIBS epitope that may play a role in the regulation of ligand-binding affinity and intracellular signaling.

Animals↗

PubMed1996

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.

Adenine Nucleotides↗

PubMed1996

One-step affinity purification of recombinant alphavbeta3 integrin from transfected cells.

Vitronectin receptor (alphavbeta3 integrin) is present on the surface of many types of cells. We describe a simple, fast, and reliable method of purification of recombinant human alphavbeta3 from Chinese hamster ovary (CHO) cells transfected with alphavbeta3 (VNRC3 cells). The method consists of two steps: lysis of the cells and affinity chromatography of the lysate on a GRGDSPK-Sepharose column. The yield of the procedure was about 79%. The purified receptor migrated as two bands on a silver stained SDS-polyacrylamide gel, corresponding to the alphav and beta3 subunits, and was recognized by monoclonal antibodies directed against alphav and the alphavbeta3 complex, but not by monoclonal antibody specific for the alphaIIbbeta3 complex. This receptor also bound to immobilized vitronectin, von Willebrand factor, and echistatin. However, binding to immobilized fibrinogen was not observed. Purified recombinant alphavbeta3 demonstrated greater immunoreactivity with LM 609, an alphavbeta3 complex-specific monoclonal antibody, than alphavbeta3 purified from placenta. As visualized by SDS-polyacrylamide gel electrophoresis, preparations of placenta-derived alphavbeta3 contained several contaminating proteins that were not present in preparations of recombinant alphavbeta3 purified from the transfected CHO cells.

Animals↗

PubMed1996

Surface-bound heparin fails to reduce thrombin formation during clinical cardiopulmonary bypass.

The hypothesis that heparin-coated perfusion circuits reduce thrombin formation and activity; fibrinolysis; and platelet, complement, and neutrophil activation was tested in 20 consecutive, randomized adults who had cardiopulmonary bypass. Twenty identical perfusion systems were used; in 10, all blood-contacting surfaces were coated with partially degraded heparin (Carmeda process; Medtronic Cardiopulmonary, Anaheim, Calif.). All patients received a 300 U/kg dose of heparin. Activated clotting times were maintained longer than 400 seconds. Cardiopulmonary bypass lasted 36 to 244 minutes. Blood samples for platelet count, platelet response to adenosine diphosphate, plasma beta-thromboglobulin, inactivated complement 3b, neutrophil elastase, fibrinopeptide A, prothrombin fragment F1.2, thrombin-antithrombin complex, tissue plasminogen activator, plasminogen activator inhibitor-1, plasmin alpha 2-antiplasmin complex, and D-dimer were obtained at these times: after heparin was given, 5 and 30 minutes after cardiopulmonary bypass was started, within 5 minutes after bypass was stopped, and 15 minutes after protamine was given. After cardiopulmonary bypass, tubing segments were analyzed for surface-adsorbed anti-thrombin, fibrinogen, factor XII, and von Willebrand factor by radioimmunoassay. Heparin-coated circuits significantly (p < 0.001) reduced platelet adhesion and maintained platelet sensitivity to adenosine diphosphate (p = 0.015), but did not reduce release of beta-thromboglobulin. There were no significant differences between groups at any time for fibrinopeptide A, prothrombin fragment F1.2, or thrombin-antithrombin complex or in the markers for fibrinolysis: D-dimer, tissue plasminogen activator, plasminogen activator inhibitor-1, and alpha 2-antiplasmin complex. In both groups, concentrations of prothrombin fragment F1.2 and thrombin-antithrombin complex increased progressively and significantly during cardiopulmonary bypass and after protamine was given. Concentrations of D-dimer, alpha 2-antiplasmin complex, and plasminogen activator inhibitor-1 also increased significantly during bypass in both groups. Fibrinopeptide A levels did not increase during bypass but in both groups increased significantly after protamine was given. No significant differences were observed between groups for levels of inactivated complement 3b or neutrophil elastase. Radioimmunoassay showed a significant increase in surface-adsorbed antithrombin on coated circuits but no significant differences between groups for other proteins. We conclude that heparin-coated circuits used with standard doses of systemic heparin reduce platelet adhesion and improve platelet function but do not produce a meaningful anticoagulant effect during clinical cardiopulmonary bypass. The data do not support the practice of reducing systemic heparin doses during cardiac operations with heparin-coated extracorporeal perfusion circuitry.

Anticoagulants↗

PubMed1996

Nafamostat mesilate, a broad spectrum protease inhibitor, modulates platelet, neutrophil and contact activation in simulated extracorporeal circulation.

Activation of humoral and cellular participants in inflammation enhances the risk of postoperative bleeding and multiple organ damage in cardiopulmonary bypass (CPB). We now compare the effects of heparin alone in combination with nafamostat mesilate (NM), a protease inhibitor with specificity of trypsin-like enzymes, in an extracorporeal circuit which simulates CPB. NM significantly inhibits the release of platelet beta-thromboglobulin (beta TG) at 60 and 120 min. Platelet counts do not differ. ADP-induced aggregation decreases in circuits with NM, which is due to a direct effect of NM on platelet function. NM prevents any significant release of neutrophil elastase; at 120 min, plasma elastase-alpha 1-antitrypsin complex is 0.16 micrograms/ml in the NM group and 1.24 micrograms/ml in the control group. NM completely inhibits formation of complexes of C1 inhibitor with kallikrein and FXIIa. NM does not alter markers of complement activation (C1-C1-inhibitor complex and C5b-9), or indicators of thrombin formation (F1.2). However, at 120 min, thrombin activity as measured by release of fibrinopeptide A is significantly decreased. The data indicate that complement activation during CPB correlates poorly with neutrophil activation and that either kallikrein or FXIIa or both may be more important agonists. The ability of NM to inhibit two important contact system proteins and platelet and neutrophil release raises the possibility of suppressing the inflammatory response during clinical CPB.

Anticoagulants↗