The effects of hydroxyethyl starches of varying molecular weights on platelet function

We evaluated the effect of various hydroxyethyl starch (HES) solutions on platelet function. Blood was obtained before and after the IV infusion (10 mL/kg) of saline (n = 10), HES 70/0.5--0.55 (molecular weight in kD/degree of substitution; n = 10), HES 130/0.38--0.45 (n = 10), HES 200/0.6--0.66 (n = 10), or HES 450/0.7--0.8 (n = 10) in otherwise healthy patients scheduled for elective surgery. Collagen and epinephrine were used as agonists for assessment of platelet function analyzer closure times. Flow cytometry was used to assess agonist-induced expression of activated glycoprotein IIb/IIIa complex and P-selectin. Infusion of HES 450/0.7--0.8, HES 200/0.6--0.66, and HES 70/0.5--0.55 prolonged closure times and reduced glycoprotein IIb/IIIa expression, whereas saline and HES 130/0.38--0.45 had no significant effect on platelet variables. P selectin expression was not affected by any solution tested. In vitro experiments demonstrated a less inhibiting effect of HES 130/0.38--0.45 on closure times when compared with other HES solutions. This study shows that HES 450/0.7--0.8, HES 200/0.6--0.66, and HES 70/0.5--0.55 inhibit platelet function by reducing the availability of the functional receptor for fibrinogen on the platelet surface. Our data indicate that fluid resuscitation with HES 130/0.38--0.45 may reduce the risk of bleeding associated with synthetic colloids of higher molecular weight and degree of substitution.     

The effect of hydroxyethyl starch 200 kD on platelet function

We evaluated the effects of hydroxyethyl starch with a molecular weight of 200 kD (HES 200 kD) on platelets to gain insight into the potential mechanisms involved in the anticoagulant effects of HES 200 kD. Blood was obtained before and after an IV infusion (10 mL/kg) of either saline (n = 15) or HES 200 kD (n = 15) in otherwise healthy patients scheduled for minor elective surgery. Flow cytometry was used to assess the expression of glycoprotein (GP) IIb-IIIa, GP Ib, and P-selectin on agonist-activated platelets. Overall platelet function was evaluated by assessing thromboelastographic maximum amplitude (MA) in celite-activated blood and platelet function analyzer-closure times by using collagen/adenosine diphosphate cartridges. Saline infusion had no effects on platelet variables, whereas HES 200 kD reduced GP IIb-IIIa expression and MA and prolonged platelet function analyzer-closure times, without affecting the expression of P-selectin and GP Ib. In vitro experiments extended these observations by a concentration-related inhibiting effect of HES 200 kD on GP IIb-IIIa expression. This study demonstrates that cellular abnormalities with decreased availability of platelet GP IIb-IIIa are involved in the anticoagulant effects of HES 200 kD.     

The GP IIb/IIIa inhibitor abciximab (ReoPro) decreases activation and interaction of platelets and leukocytes during in vitro cardiopulmonary bypass simulation.

OBJECTIVE: Cardiopulmonary bypass (CPB) induces a systemic inflammatory response and increases expression of the platelet activation marker P-selectin which mediates binding of platelets to leukocytes. Inhibition of the platelet GP IIb/IIIa receptor during CPB has been shown to protect platelets without increasing bleeding complications and is assumed to reduce the inflammatory response. The aim of this study was to investigate the effect of the GP IIb/IIIa inhibitor abciximab (ReoPro) on the function and interaction of platelets and leukocytes during experimental CPB. METHODS: Heparinized (3 U/ml) fresh whole blood of healthy volunteers was treated before continuous in vitro circulation in a well established CPB model with 3.2 microg/ml abciximab (n=6) or left untreated as control (n=6). Measurements were made before (baseline) and after 30 and 60 min of circulation and comprised: percentage of platelets expressing P-selectin and percentage of platelet-bound leukocytes (flow cytometry), release of the leukocyte activation marker PMN-elastase (ELISA), and platelet and leukocyte counts. RESULTS: Abciximab almost completely prevented a CPB-induced increase of platelet P-selectin and platelet-leukocyte binding after 30 and 60 min of circulation, and significantly inhibited release of PMN-elastase after 30 min of circulation. Furthermore, abciximab significantly inhibited a CPB-induced decrease of platelet and leukocyte counts. CONCLUSIONS: Abciximab inhibits CPB-induced activation, interaction and consumption of platelets and leukocytes in vitro. GP IIb/IIIa inhibition should be considered as a promising approach not only to conserve platelet function but also to inhibit pro-inflammatory events during CPB in vivo.     

Platelet P-selectin and GPIIb/IIIa expression after liver transplantation and resection

Platelet dysfunction contributes to haemostatic defects, possibly leading to bleeding complications. We hypothesised that liver transplantation and liver resection, together with portal clamping time, might be a potential stimulus for platelet activation. Therefore, we determined the expression of platelet GPIIb/IIIa and P-selectin, representing important platelet activation markers, and the thrombopoietin (TPO) serum level after transplantation and resection. Twenty patients [ten that had undergone orthotopic liver transplantation (OLT), ten with liver resection (LRX)] were included in the study. From sequential venous blood samples, surface expression of GPIIb/IIIa and P-selectin was quantified by flow cytometry, and TPO serum levels were determined by ELISA. Baseline GPIIb/IIIa receptor expression on circulating platelets was significantly reduced in the OLT group compared to the LRX group and healthy volunteers. GPIIb/IIIa expression after activation with TRAP-6 increased significantly (P<0.001) in the LRX group but not in the OLT group. P-selectin expression after TRAP-6 stimulation increased significantly (P<0.001) in the LRX group, being comparable to that in healthy volunteers, whereas only a very low increase in the OLT group was found. In the OLT group, TPO serum levels were in the lower normal range and rose above the upper limit of normal values 24 h after reperfusion. These data indicate that neither liver transplantation nor liver resection influences GPIIb/IIIa and P-selectin expression on circulating platelets. There was a lack of expression in cirrhotic patients and unimpaired baseline expression and functional reserve in non-cirrhotic liver-resection patients. After liver transplantation, increasing serum TPO levels, which indicated a recovering graft function, resulted in rising peripheral platelet counts.     

Epinephrine enhances platelet-neutrophil adhesion in whole blood in vitro

Previous studies showed that alpha- or beta-adrenoceptor stimulation by catecholamines influenced neutrophil function, cytokine liberation, and platelet aggregability. We investigated whether adrenergic stimulation with epinephrine also alters platelet-neutrophil adhesion. This might be of specific interest in the critically ill, because the increased association of platelets and neutrophils has been shown to be of key importance in inflammation and thrombosis. For this purpose, whole blood was incubated with increasing concentrations of epinephrine (10 nM, 100 nM, and 1 microM). To distinguish receptor-specific effects, a subset of samples was incubated with propranolol (10 microM) or phentolamine (10 microM) before exposure to epinephrine. After incubation, another subset of samples was also stimulated with 100 nM of N-formyl-methionyl-leucyl-phenylalanine. All samples were stained, and platelet-neutrophil adhesion and CD45, L-selectin, CD11b, P-selectin glycoprotein ligand-1, glycoprotein IIb/IIIa, and P-selectin expression were measured by two-color flow cytometry. Epinephrine significantly enhanced platelet-neutrophil adhesion and P-selectin and glycoprotein IIb/IIIa expression on platelets. CD11b and L-selectin expression on unstimulated neutrophils remained unchanged, whereas N-formyl-methionyl-leucyl-phenylalanine-induced upregulation of CD11b and downregulation of L-selectin were suppressed by epinephrine. beta-Adrenergic blockade before incubation with epinephrine increased platelet-neutrophil aggregates and adhesion molecule expression (CD11b, P-selectin, and glycoprotein IIb/IIIa) even further. These results demonstrate that epinephrine enhances platelet-neutrophil adhesion. The alpha-adrenergic receptor-mediated increase in P-selectin and glycoprotein IIb/IIIa expression on platelets may contribute substantially to this effect. Our study shows that inotropic support enhances the platelet-neutrophil interaction, which might be crucial for critically ill patients.     

Pharmacologic platelet anesthesia by glycoprotein IIb/IIIa complex antagonist and argatroban during in vitro extracorporeal circulation

OBJECTIVE: Contact between blood and the synthetic surfaces of a cardiopulmonary bypass circuit leads to platelet activation, and resultant platelet dysfunction contributes to postoperative bleeding. We compared the effects of various platelet inhibitors on preservation of platelet function during simulated cardiopulmonary bypass circulation. METHODS: Fresh human blood was recirculated in an in vitro cardiopulmonary bypass model circuit. We measured various platelet activation markers including expressions of PAC-1 and P-selectin, annexin V binding, and microparticle formations by means of whole-blood flow cytometry. RESULTS: Two types of glycoprotein IIb/IIIa complex antagonists, peptide-mimetic FK633 and abciximab and prostaglandin E(1), significantly prevented platelet loss and the increase in binding of PAC-1, an antibody specific for fibrinogen receptor on activated platelets, during extracorporeal circulation of heparinized blood. These antagonists significantly suppressed but did not abolish P-selectin expression, annexin V binding, and microparticle formation. Anti-von Willebrand factor monoclonal antibody and aurin tricarboxylic acid (an inhibitor of glycoprotein Ib) had no effect on platelet activation during simulated cardiopulmonary bypass circulation. These data suggest that inhibition of fibrinogen binding glycoprotein IIb/IIIa complex is partly effective in attenuating platelet activation in a heparinized cardiopulmonary bypass model circuit. The direct thrombin inhibitor argatroban prevented platelet loss and expression of P-selectin significantly more than did heparin. A combination of FK633 with argatroban as a substitute for heparin further prevented platelet loss and platelet secretion during simulated cardiopulmonary bypass circulation, although the inhibition of microparticle formation was less. CONCLUSION: The inhibition of both platelet adhesion and thrombin may be effective to preserve platelet number and function during cardiopulmonary bypass circulation.     

Reduced P-selectin expression on circulating platelets after prolonged cold preservation in renal transplantation

The uremic state in patients with terminal renal insufficiency is accompanied by a bleeding tendency connected with platelet dysfunction. Prolonged cold ischemia and inflammatory interactions between leukocytes, platelets and endothelial cells contribute to ischemia-/reperfusion (I/R) injury and may impair long-term graft survival. We evaluated the influence of the duration of cold preservation time on the expression of platelet GPIIb/IIIa and P-selectin and on the formation of leukocyte-platelet complexes after kidney transplantation. Fourteen patients undergoing kidney transplantation were divided into group I with long preservation time (26.6 +/- 1.9 h) and group II with short preservation time (8 +/- 6.1 h). Five venous blood samples (3 ml) were taken before induction of anesthesia, 12 h, 2, 7 and 14 d after transplantation. Surface expression of the GPIIb/IIIa, P-selectin and the percentage of platelet-granulocyte complexes were quantified by flow cytometry. Additionally blood from seven healthy volunteers was analyzed. GPIIb/IIIa and P-selectin expression on circulating platelets were significantly decreased in the long and the short-term graft preservation group compared with healthy volunteers. A significantly reduced P-selectin expression was found in the long-term preservation group compared with the short-term group. The percentage of platelet-granulocyte complexes also decreased in both preservation groups in the first 2 d after reperfusion and remained in this state in the long-term preservation group. Reduced expression of P-selectin on circulating platelets may be an indicator of I/R injury after prolonged kidney graft preservation.     

Effects of two different hydroxyethyl starch solutions (HES200/0.5 vs. HES130/0.4) on the expression of platelet membrane glycoprotein

BACKGROUND: There are various hydroxyethyl starch (HES) solutions with different degrees of hydroxylation and different molecular weights. HES200/0.5 solution is most commonly used. HES130/0.4 is a new HES solution and is the 'state-of-the-art' in volume substitution. However, the mechanism of the observed anticoagulation action of HES has not been fully delineated. The objective of this study was to further investigate the effect of HES200/0.5 and HES130/0.4 on platelet coagulation. METHODS: Sixty ASA I-II patients undergoing elective minor surgery were randomly allocated to receive an intravenous infusion (20 ml/kg) of lactated Ringer's solution (group L), HES200/0.5 (group H) or HES130/0.4 (group V) after the induction of anesthesia. The expression of CD42b, CD41/61 and CD62p in vivo was assessed on non-stimulated platelets and adenosine diphosphate (ADP) agonist-activated platelets using flow cytometry. RESULTS: Resting glycoprotein expression of the non-stimulated platelets was observed. HES200/0.5 and HES130/0.4 reduced the CD42b, CD41/61 and CD62p expression of ADP-agonist-activated platelets at 15 min after intravenous infusion. At 6 h after intravenous infusion, the trend of decreasing expression of activated CD42b, CD41/61 and CD62p was maintained in group H. However, CD42b, CD41/61 and CD62p expression returned to the pre-operative level in group V. CONCLUSION: This study showed that both HES200/0.5 and HES130/0.4 can inhibit platelet coagulation. Platelet dysfunction experienced a faster recovery after the infusion of HES130/0.4 than after HES200/0.5. Liquid resuscitation with HES130/0.4 may decrease the risk of hemorrhage in the operative period.     

The effect of intravenously administered magnesium on platelet function in patients after cardiac surgery.

After cardiac surgery, magnesium is often administered for prophylaxis and treatment of cardiac arrhythmias. Magnesium, however, inhibits platelet function in vitro and in healthy volunteers. We performed a randomized, blinded, and placebo-controlled study to investigate the effect of magnesium on platelet function in patients after cardiac surgery. We studied patients who underwent uneventful coronary revascularization with cardiopulmonary bypass on the first postoperative day. Before and after an infusion of either 5.4 mmol magnesium (n = 19) or saline (n = 20), platelet function was investigated by means of in vitro bleeding time, platelet aggregation, and flow-cytometric assays. In addition, to investigate platelet function in vitro, 1, 5, and 10 mM magnesium were added to platelet-rich plasma before and 24 h after surgery in 30 patients. Compared with the control group, magnesium prolonged the in vitro bleeding time (22%) and inhibited ADP- and collagen-induced platelet aggregation (13% and 17%), platelet P-selectin expression (18%), and the binding of fibrinogen to the platelet glycoprotein IIb/IIIa receptor (10%). Magnesium also led to significant dose-dependent inhibition of platelet aggregation (19%), P-selectin expression (14%), and fibrinogen binding (11%) before and after surgery in vitro. Although the antithrombotic effect of magnesium may be beneficial in patients after coronary revascularization, large-dose magnesium therapy should be carefully considered in patients with impaired platelet function and co-existing bleeding disorders. IMPLICATIONS: In a randomized, blinded, placebo-controlled study of patients 24 h after coronary artery bypass grafting, IV administered magnesium inhibited platelet function in vitro and in vivo.     

The effects of aprotonin on platelets in vitro using whole blood flow cytometry

We sought to evaluate the effects of aprotinin on the number and function of the platelet glycoprotein (GP) IIb-IIIa receptor and on the expression of P-selectin in vitro in order to gain insight into the potential mechanisms involved in the platelet-protective action of aprotinin during cardiopulmonary bypass. Aprotinin at 50 to 200 kallikrein inhibiting units/mL decreased the expression of activated GP IIb-IIIa complex in response to adenosine diphosphate or thrombin receptor activator peptide 6 in a dose-dependent manner in both citrated and heparinized whole blood experiments. Aprotinin inhibited adenosine diphosphate-induced platelet aggregation, but it exhibited no effect on the expression of GP IIIa and P-selectin. These results indicate that aprotinin interferes with the platelet fibrinogen receptor function during pharmacological activation. Reduced aggregability and platelet adhesion to fibrinogen adsorbed to synthetic surfaces in the presence of aprotinin may prevent platelet consumption during clinical cardiopulmonary bypass. This in vitro study demonstrates that aprotinin decreases the agonist-induced expression of activated GP IIb-IIIa receptors that play a major role in platelet aggregation and adhesion to biomaterial surfaces. IMPLICATIONS: This in vitro study demonstrates that aprotinin decreases the agonist-induced expression of activated glycoprotein IIb-IIIa receptors that play a major role in platelet aggregation and adhesion to biomaterial surfaces.     

Modulation of platelet surface adhesion receptors during cardiopulmonary bypass.

Alterations in platelet receptors critical to adhesion may play a role in the pathogenesis of the qualitative platelet defect associated with cardiopulmonary bypass. Using flow cytometry, we measured changes in the following platelet surface adhesive proteins: the von Willebrand factor receptor, glycoprotein Ib; the fibrinogen receptor, glycoprotein IIb/IIIa; the thrombospondin receptor, glycoprotein IV; the adhesive glycoprotein granule membrane protein 140, whose expression also reflects platelet activation and alpha-granule release; and, as a control, the nonreceptor protein HLA, A,B,C. Glycoprotein Ib decreased during cardiopulmonary bypass (P less than 0.05) and reached a nadir at 72% (P less than 0.05) of its baseline value at 2-4 h after bypass. This decrease correlated (r = 0.76) with the magnitude of platelet activation (alpha-granule release) in any given patient, but even platelets that were not activated demonstrated a decrease in glycoprotein Ib expression. Glycoprotein IIb/IIIa also decreased in both the activated (47% of baseline, P less than 0.01) and unactivated (63% of baseline, P less than 0.01) subsets of platelets at the end of cardiopulmonary bypass. Glycoprotein IV and HLA A,B,C did not decrease, but instead increased 2-4 h after cardiopulmonary bypass (P less than 0.05). We conclude that cardiopulmonary bypass produces selective decreases in surface glycoproteins Ib and IIb/IIIa as well as in platelet activation; that these two alterations are temporally but not necessarily mechanistically linked; and that these changes have the potential to adversely affect platelet function.     

Combined administration of nitric oxide gas and iloprost during cardiopulmonary bypass reduces platelet dysfunction: a pilot clinical study

BACKGROUND: Thrombocytopenia and platelet dysfunction are major mechanisms of cardiopulmonary bypass-induced postoperative hemorrhage. This study evaluated the effects of low amounts of nitric oxide, iloprost (prostacyclin analog), and their combination administered directly into the oxygenator on platelet function, platelet-leukocyte interactions, and postoperative blood loss in patients undergoing coronary artery bypass grafting. METHODS: Blood samples from 41 patients randomized to the control, nitric oxide (20 ppm), iloprost (2 ng x kg -1 x min -1 ), or nitric oxide plus iloprost groups were collected during cardiopulmonary bypass. Platelets and leukocytes were enumerated. Platelet membrane glycoprotein Ib and glycoprotein IIb/IIIa, P-selectin, platelet-derived microparticles, leukocyte CD11b/CD18 (Mac-1), and platelet-leukocyte aggregate were quantified by means of flow cytometry. Collagen and thrombin receptor-activating peptide-induced platelet aggregation in whole blood was analyzed by means of aggregometry. RESULTS: Both nitric oxide or iloprost attenuated cardiopulmonary bypass-induced thrombocytopenia, reduction of glycoprotein Ib and glycoprotein IIb levels, translocation of P-selectin, microparticle formation, Mac-1 upregulation, and suppression of collagen-induced aggregation. Nitric oxide plus iloprost was significantly more effective in preventing thrombocytopenia, microparticle formation, and P-selectin translocation. Moreover, this treatment preserved thrombin receptor-activating peptide-induced aggregation, which was not rescued by single treatments. Both nitric oxide and nitric oxide plus iloprost attenuated postoperative blood loss. CONCLUSIONS: Nitric oxide plus iloprost reduced the deleterious effects of cardiopulmonary bypass, such as thrombocytopenia, platelet activation, platelet-leukocyte aggregate formation, and suppression of platelet aggregative responses. The reduced postoperative bleeding observed with this treatment suggests that this is a new and clinically feasible therapeutic option for patients subjected to cardiopulmonary bypass.     

Platelet glycoprotein IIb/IIIa receptor antagonist (abciximab) inhibited platelet activation and promoted skin flap survival after ischemia/reperfusion injury

BACKGROUND: Evidence has shown that platelets play an important role in the pathogenesis of flap failure. Employing a rat inferior epigastric artery skin flap as a flap reperfusion injury model, we investigated whether platelet activation was involved in the skin flap failure and whether administration of abciximab (ReoPro, chimeric 7E3 Fab) could decrease platelet activation/aggregation and promote flap survival. METHODS: Normal saline and abciximab (0.06 mg/kg; 0.2 mg/kg; 1 mg/kg) were injected intravenously into skin flaps 30 min before reperfusion and 1 h after reperfusion (each subgroup n = 6). Platelet activation as demonstrated by P-selectin (CD62P) was analyzed by flow cytometry. P-selectin expression on flap vessels was detected by immunohistochemical staining. Platelet aggregation was induced with adenosine diphosphate (ADP). Laser Doppler flowmetry monitored tissue perfusion. The surviving area was evaluated 7 days postoperatively. RESULTS: CD62P progressively increased after reperfusion. The peak CD62P occurred after reperfusion for 12 h. Immunohistochemical staining showed CD62P significantly deposited on the endothelium after reperfusion. Administration of abciximab (1 mg/kg) effectively improved flap survival rate (P = 0.003), significantly decreased ADP-induced platelet aggregation (P < 0.001), and suppressed CD62P expression on blood platelets (P = 0.002) and its deposition on the flap vessels. CONCLUSION: Abciximab promotion of skin flap survival is due to blocked platelet activation/aggregation and decreased activated-platelet deposition on the vascular endothelium. Thus, administration of a platelet glycoprotein IIb/IIIa receptor antagonist such as abciximab may save the skin flap from reperfusion injury after a long period of ischemia.     

New RGD analogue inhibits human platelet adhesion and aggregation and eliminates platelet deposition on canine vascular grafts.

Platelet adhesion and aggregation are mediated by fibrinogen via the receptor glycoprotein IIb/IIIa, which recognizes the arginine-glycine-aspartic (RGD) amino-acid sequence. We investigated the ability of 8-guanidino-octanoyl-Asp-Phe (SC-49992), an intravenously infused, stable RGD analogue, to inhibit human platelet function in vitro and to reduce in vivo canine platelet deposition on prosthetic grafts. Human platelet aggregation induced by 10 mumol/L adenosine diphosphate was inhibited in a concentration dependent manner with an ED50 of 1 microgram/ml of SC-49992. Adenosine diphosphate-induced secretion, which is dependent on fibrinogen occupancy of the glycoprotein IIb/IIIa receptor, was reduced in a concentration dependent manner, also with an ED50 of 1 microgram/ml. Thrombin-induced secretion, which is independent of fibrinogen binding, was unaffected. Activation-dependent platelet adhesion to fibrinogen substrates was reduced in a concentration-dependent manner by SC-49992. Platelet adhesion to fibronectin substrates was also reduced by the analogue, but to a lesser extent. SC-49992 effectively eluted glycoprotein IIb/IIIa bound to RGD derivatized sepharose. Eight thrombosis-prone dogs had polytetrafluoroethylene femoral artery grafts placed. Dogs received the RGD analogue or a normal saline infusion during their first graft procedure. One week later a second contralateral femoral graft with infusion of the other agent was performed. Aggregometry during RGD analogue infusion demonstrated inhibition of induced aggregation, whereas normal saline infusion had no effect. As measured by the adherence of platelets labeled with indium III 8-guanidino-octanoyl-Asp-Phe reduced platelet deposition on vascular grafts by more than 90% (p = 0.0006, log transformed data, paired t test.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of laser welding with human serum albumin on the expression of P-selectin on platelets

BACKGROUND AND OBJECTIVE: Artery repair by means of laser energy induces activation of platelets with a risk of thrombosis and local inflammatory reactions. The aim of this study was to investigate the effect of human serum albumin, the most common solder in laser surgery, on platelet activation. STUDY DESIGN/MATERIALS AND METHODS: Platelet activation was evaluated in canine blood by using two-color flow cytometry with a phycoerythrin-labeled antibody to a common platelet marker, glycoprotein IIb/IIIa and fluorescein isothiocyanate-labeled antibody to a platelet activation molecule, P-selectin. Human serum albumin was applied in vitro and in vivo, as a solder during laser reconstruction of canine arteries. RESULTS: In vitro, albumin significantly (P < 0.01) reduces the expression of P-selectin on platelets. This is most likely related to the blockage of P-selectin by albumin, which binds to the platelet surface, as confirmed by flow cytometry with fluorescein isothiocyanate-labeled albumin. In vivo, application of albumin solder tended to result in a lower percentage of P-selectin-expressing platelets in laser-repaired arteries compared to suture-repaired arteries. CONCLUSION: Albumin decreases the percentage of P-selectin-expressing platelets in vitro. Further research may allow the platelet activation inhibiting properties of albumin to be further optimized in vivo.     

Human neutrophil cathepsin G is a potent platelet activator

Purpose: Neutrophil activation has been implicated in the pathophysiologic condition of ischemia-reperfusion injury, the formation of arterial aneurysms, the progression of myocardial ischemia, and the initiation of deep venous thrombosis. Activated neutrophils release cathepsin G, a serine protease, from their granules, which may cause platelet activation that leads to intravascular thrombosis, tissue infarction, and systemic release of the thrombogenic products of platelet granules. This study used flow cytometry to quantify the extent of cathepsin G–induced platelet activation and degranulation through changes in the expression of platelet surface glycoproteins.Methods: Increasing concentrations of human neutrophil–derived cathepsin G were incubated with washed platelets or whole blood from healthy human donors. The platelet surface expression of glycoproteins, including P-selectin, a platelet membrane glycoprotein only expressed after platelet alpha granule release, were determined by quantifying the platelet binding of a panel of fluorescently labeled monoclonal antibodies. Results were compared with the effect of a maximal dose of thrombin, the most potent known platelet activator.Results: In a washed platelet system, cathepsin G increased platelet surface expression of P-selectin (an activation-dependent neutrophil binding site), the glycoprotein IIb/IIIa complex (fibrinogen receptor), and glycoprotein IV (thrombospondin receptor), and decreased surface expression of glycoprotein Ib (von Willebrand factor receptor) to an extent comparable to maximal thrombin. However, these effects were not observed in a whole blood system. Further experiments revealed that preexposure to plasma completely inhibited cathepsin G–induced washed platelet activation and degranulation. Prostacyclin treatment of washed platelets markedly inhibited cathepsin G–induced platelet activation.Conclusions: Cathepsin G is a very potent platelet agonist and degranulator, comparable to maximal thrombin, which alters platelet surface glycoprotein expression for enhanced neutrophil binding and effective platelet aggregation. This study helps to elucidate a possible pathway through which neutrophils may directly activate platelets, leading to intravascular thrombosis, irreversible ischemia, and tissue death in cardiovascular disease states. Patients with diseased endothelium that is deficient in prostacyclin production may be particularly prone to the detrimental effects of neutrophil-derived cathepsin G platelet activation. (J VASC SURG 1994;19:306-19.)     

Glycoprotein IIb/IIIa receptor inhibitor attenuates platelet aggregation induced by thromboxane A2 during in vitro nonpulsatile ventricular assist circulation

A recent development in antithrombotic research allows the inhibition of platelet aggregation via protection of the glycoprotein IIb/IIIa receptor on the platelet membrane. We hypothesized that a GP IIb/IIIa receptor inhibitor would inhibit thromboxane-induced platelet aggregation during circulation in our in vitro ventricular assist device (VAD) circuit and preserve long-term platelet function. Twenty-one in vitro nonpulsatile centrifugal VAD circuits were simulated for 4 days using 450 ml of fresh human whole blood with or without glycoprotein IIb/IIIa receptor inhibitor (tirofiban). Platelet aggregation and degranulation were measured in whole blood induced by ristocetin, collagen, ADP, and thromboxane A2 (TXA2). The tirofiban-treated group preserved the platelet count and tended to exert these beneficial effects by inhibiting pathologic platelet aggregation induced by TXA2, collagen, and ADP as well as degranulation. Tirofiban may be useful in preserving platelet number and function during clinical VAD use.     

比较不同分子量羟乙基淀粉对术中血液循环及凝血功能的影响

目的多中心、双盲、平行对照比较术中输注国产6%羟乙基淀粉200/0.5(6%HES200/0.5,盈源)和6%羟乙基淀粉130/0.4(6%HES130/0.4,万汶)对术中血流动力学、血液流变学及凝血功能的影响。方法150例择期手术的患者随机均分为Y组和V组。手术开始后,Y组输注6%HES200/0.51000ml,V组输注6%HES130/0.41000ml。分别于麻醉前(T0)、输注开始时(T1)、输注开始后30min(T2)、60min(T3)、90min(T4)、120min(T5)各时点监测患者血流动力学参数,在麻醉前及输注结束后10min抽血检测凝血功能及血液流变学参数。结果与T0时相比,两组患者T1时MAP均明显下降(P<0.05),V组T4、T5时MAP明显降低(P<0.05);T3时V组HR增快(P<0.05),输液结束后两组凝血酶原时间(PT)及部分凝血酶原时间(APTT)均有所延长(P<0.05),纤维蛋白原下降(FIB)(P<0.05),血小板计数(Plt)减少(P<0.05),但两组组间差异无统计学意义;两组液体均可降低全血高、低切变率(P<0.05),对血浆粘度无明显作用。两组术中输血量及晶体输入量差异无明显统计学意义。结论输入6%HES200/0.5或6%HES130/0.41000ml均可有效维持血流动力学稳定,改善机体微循环,但对凝血功能均有尚可耐受的影响。     

In vitro effects of gelatin solutions on platelet function: a comparison with hydroxyethyl starch solutions

The aim of this study was to investigate the effects of various gelatin and hydroxyethyl starch solutions on platelet reactivity. Citrated whole blood was obtained from 20 healthy volunteers. Expression of glycoprotein (GP) IIb-IIIa and p-selectin were determined using whole blood flow cytometry on both resting and agonist-activated platelets before and after in vitro haemodilution (20% and 40%) using oxypolygelatin, modified gelatin, urea-linked gelatin, hydroxyethyl starch (HES) 130 (mean molecular weight in kDa), HES 200, HES 450 and HES 550. High degrees of haemodilution using oxypolygelatin had no significant effect, similar to HES 130, whereas modified gelatin inhibited GP IIb-IIIa expression, similar to HES 200 and HES 450. Urea-linked gelatin significantly increased the expression of GP IIb-IIIa, similar to HES 550. p-selectin expression remained unchanged in all samples. The present in vitro study indicates that chemical characteristics of colloidal solutions modulate their influence on platelet reactivity.     

羟乙基淀粉对血液流变学及凝血功能的影响

目的 观察输注6％羟乙基淀粉（HES200／0．5）对病人血液流变学及凝血功能的影响。方法 30例硬膜外阻滞下手术病人,在硬膜外阻滞前输注贺斯12ml/kg。输液前后分别抽取静脉血测定：（1）全血低切、中切、高切粘度,红细胞比容,全血低切、中切、高切还原粘度,血沉,红细胞聚集指数、刚性指数、变形指数。（2）血小板计数（PLC）,血小板粘附率（PAD）,血小板1分钟聚集率（PAG1）、3分钟聚集率（PAG3）和最大聚集率（PAGM）。（3）凝血酶原时间（PT）,凝血酶时间（TT）,部分凝血活酶时间（APTT）和纤维蛋白原浓度（Fib)。结果 输液后全血粘度高切变率、中切变率明显下降,全血还原粘度高切变率、血沉、红细胞刚性指数在输液后显著降低,血小板粘附及聚集功能、TT、APTT及Fib输液后差异无显著意义,只有PT显著延长。结论 静注500－1000ml6%HES,可改善血液流变性,对凝血功能无影响。