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.     

In Vivo Evaluation of Platelet Activation by Different Cellulosic Membranes

Abstract: This study was undertaken to evaluate platelet activation in vivo induced by different cellulosic membranes by measuring the expression of P-selectin on the platelet surface during hemodialysis in 9 uremic patients. Hollow fiber dialyzers of similar surface with different cellulosic membranes (Cuprophan, cellulose acetate, cellulose triacetate, and Hemophan) were evaluated and compared to a synthetic membrane (polysulfone). Blood samples were obtained before hemodialysis and from the efferent and afferent limbs 5 min after the beginning of dialysis. P-select in exposure was evaluated by flow cytometry (FACScan) using a monoclonal antibody (RUU 2.17). The percentage of platelets expressing P-select in before hemodialysis and the percentage from the arterial line during hemodialysis were similar. All membranes evaluated induced platelet activation (estimated as the increase in percentage of platelets expressing P-selectin in samples obtained from the venous line with respect to the arterial line). Cuprophan induced more platelet activation than any other membrane (p < 0.05). The activation induced by cellulose acetate and cellulose triacetate membranes was also higher than that observed with Hemophan (p < 0.05). Hemophan-induced platelet activation was similar to that of polysulfone. These results indicate that all cellulosic membranes induce platelet activation during hemodialysis although there are quantitative differences among them. While Cuprophan induced the highest degree of platelet activation, Hemophan was the only cellulosic membrane that showed a degree of platelet activation similar to the biocompatible membrane polysulfone.     

The effects of heparin, protamine, and heparinase 1 on platelets in vitro using whole blood flow cytometry

The effects of heparinization and the reversal of heparin activity on platelet function after cardiopulmonary bypass have not been well defined. Flow cytometry has become a convenient and powerful technique for characterizing platelets. We examined the expression of a secretion marker (P-selectin) and an aggregation marker (activated fibrinogen receptor GP IIb-IIIa) on normal platelets in response to heparin, heparinase 1, and protamine in vitro using whole blood flow cytometry. Unfractionated heparin increased adenosine diphosphate-induced expression of P-selectin and GP IIb-IIIa in a dose-dependent manner. Heparinase 1 alone decreased both markers of platelet activation. Protamine alone increased P-selectin expression but had no effect on GP IIb-IIIa expression. Heparinase 1 antagonized the stimulatory effect of heparin on both markers. In contrast, protamine antagonized the effect of heparin on GP IIb-IIIa expression but potentiated the effect of heparin on P-selectin expression. These in vitro observations suggest that 1) both heparin and its reversal agents affect platelet secretion and aggregation, and 2) heparinase 1 reverses heparin-induced platelet preactivation more effectively than protamine. IMPLICATIONS: This experimental in vitro study demonstrates that heparin and its reversal agents affect platelet secretion and aggregation.     

Platelet function and anesthetics in cardiac surgery: an in vitro and ex vivo study.

We studied the effects of the anesthetics commonly used in cardiac surgery on platelet function. Fentanyl, droperidol, succinylcholine, pancuronium, thiopental, and diazepam at therapeutic concentrations were tested for their in vitro effects on the expression of platelet membrane glycoproteins Ib and IIbIIIa (GpIb, GpIIb-IIIa) and of P-selectin in anticoagulated whole blood by flow cytometry. The expression of P-selectin was determined under basal conditions, after the incubation of blood with adenosine diphosphate (ADP) 10 micromol/L, and the stable prostaglandin endoperoxide analog U46619 1 micromol/L. No drug affected the expression of P-selectin in unstimulated and ADP- or U46619-stimulated platelets, with the exception of thiopental, which markedly decreased the U46619-induced expression of P-selectin. Thiopental concentration-dependently inhibited U46619-induced and ADP-induced platelet aggregation, with effects on U46619-induced aggregation at therapeutic concentrations. To assess ex vivo effects, the same platelet markers were also assessed in blood obtained from 10 patients undergoing elective coronary surgery. Compared with basal values, platelet response to U46619 was significantly reduced just after the administration of anesthetic drugs, and the effect persisted for 48 h after surgery. Our study suggests that, at therapeutic concentrations, thiopental inhibits U46619-induced platelet activation both in vitro and ex vivo. The mechanisms responsible of this effect, together with its clinical significance, require further investigation. IMPLICATIONS: Thiopental inhibited prostaglandin-induced platelet activation at therapeutic concentrations both in vitro and ex vivo in cardiac surgical patients whereas adenosine diphosphate-induced activation was affected only at supratherapeutic drug concentrations. Thus, administration of sodium thiopental may contribute to the in vivo impairment of platelet function in patients undergoing elective cardiac surgery.     

Carotid artery anastomosis with albumin solder and near infrared lasers: a comparative study.

BACKGROUND AND OBJECTIVE: Laser tissue-welding has been used for anastomosis of carotid arteries. During welding, thermal injury sustained by the vessel walls should be minimized to prevent thrombosis. Two different types of lasers were used and effects on tissue damage were studied in vitro and in vivo. STUDY DESIGN/MATERIALS AND METHODS: End-to-end anastomosis of dog carotid arteries (n = 10) was performed by using a human albumin solder (HAS) in conjunction with Nd:YAG or diode lasers (lambda = 1.32 microm and 1.9 microm, respectively). The arteries were evaluated for patency and evidence of histologic injury after 21 days. Another group of arteries was laser soldered in vitro to measure the intimal and adventitial temperatures by using thermocouples. RESULTS: The arteries repaired with the diode laser sustained significantly less thermal damage than those repaired with Nd:YAG laser, both in vitro and in vivo. In particular, the intimal temperature was significantly lower (P < 0.05) for the diode than for the Nd:YAG repairs (approximately 35 degrees C and approximately 50 degrees C, respectively). In the latter group, the patency rate was 75%, but thrombosis occurred in 75% of the specimens at 21 days. All diode anastomoses were patent and thrombosis developed in only 17% of the arteries. CONCLUSION: Use of the diode laser and albumin solders may provide a means to successfully repair carotid arteries with minimal thermal damage.     

Platelet activation markers in patients with nephrotic syndrome. A comparative study of different platelet function tests

BACKGROUND/AIM: Enhanced platelet reactivity may play a significant role in the genesis of the hypercoagulable state of nephrotic syndrome. However, the role of platelet function testing in nephrosis is controversial, partly because the methods used to assess platelet function (platelet aggregation and immunoassays of plasma beta-thromboglobulin and platelet factor 4) have such marked methodological problems. In the present study, we evaluated several tests assessing platelet function in 18 adult patients with idiopathic nephrotic syndrome and normal renal function. METHODS: Platelet function was assessed by measurement of plasma beta-thromboglobulin (enzyme-linked immunosorbent assay, ELISA), plasma P-selectin (ELISA), circulating platelets exposing the activation-dependent antigens P-selectin (CD62P) and lysosomal GP53 (CD63) (flow cytometry), and by aggregation response to agonists such as ADP and collagen. Results were compared to those obtained in a group of 16 age- and gender-matched healthy subjects. RESULTS: Levels of plasma beta-thromboglobulin (p = 0.001), plasma P-selectin (p < 0.001), and CD62P/CD63-positive platelets (p < 0.001 for both) were increased in nephrotic patients as compared to healthy controls. Platelet hyperaggregability in vitro was found in 13/18 patients. The reproducibility of platelet activation markers, as assessed by blood sample collection a week later from all patients, was found to be higher for plasma P-selectin (Spearman correlation coefficient, R = 0.99) and circulating activated platelets (CD62P: R = 0.97; CD63: R = 0.96) than for plasma beta-thromboglobulin (R = 0.78). CONCLUSIONS: Pronounced platelet activation takes place in nephrotic syndrome and may contribute to the hypercoagulability of nephrosis. Whole blood flow cytometry assay of platelet activation and plasma P-selectin assay may represent useful tests to assess the hypercoagulable state in nephrotic 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.     

The effects of platelet apheresis in total hip replacement surgery on platelet activation

BACKGROUND: Autologous platelet rich plasma (PRP) harvest with autotransfusion devices has been used for 10 years in cardiac surgery and recently in orthopedics as a blood saving method. The quality of the harvested platelets has not been adequately examined, in part because of methodological difficulties in studying platelet function during surgery. METHODS: Twenty patients undergoing primary total hip replacement (THR) were studied. Ten patients underwent an immediate preoperative platelet apheresis to obtain concentrated platelet rich plasma (c-PRP). The other 10 patients not undergoing apheresis were allocated to a control group. Platelet activation was evaluated as the population expressing P-selectin on the surface of platelets in the c-PRP and in blood samples collected pre-, per- and postoperatively. The method used was flow cytometry. RESULTS AND CONCLUSIONS: A minor population of activated platelets was found to be circulating in the patients' blood, with a highly significant difference between patients (P = 0.005), and with a range of 1-23% in peroperative activation. PRP harvest did not significantly alter platelet activity. The platelet apheresis procedure did not inhibit platelet function in the c-PRP, as judged by a high proportion of platelets that could be activated in ADP stimulation experiments (mean value +/- SD 86% +/- 7.5%).     

The association of in vitro arachidonic acid responsiveness and plasma thromboxane levels with early platelet deposition on the luminal surface of small-diameter grafts

The response of canine platelets to arachidonic acid (AA) stimulation was studied as a predictor of thrombotic potential. Fifty mongrel dogs underwent in vitro platelet aggregation studies with adenosine diphosphate (ADP), collagen, and AA used as inducing agents. Thirty-two dogs were selected on the basis of their response to AA stimulation. Platelet aggregation in response to AA stimulation occurred in 16 (responders) and 16 showed no aggregatory response (nonresponders). The animals were divided into four groups. Group I received no antiplatelet agents (control); group II received U-63,557A, a specific thromboxane synthetase inhibitor (TSI); group III received aspirin; and group IV received aspirin and TSI. Polytetrafluoroethylene grafts were implanted in the carotid and femoral arteries of the dogs in all four groups. Plasma thromboxane (TxB₂) levels were drawn before drug treatment and 4 weeks after surgery. Platelet deposition on the luminal surface of the implanted grafts was studied in vivo with a technique that uses both ¹¹¹In-labeled platelets and ^99mTc-labeled red blood cells and was expressed as percentage of indium excess (%IE). Group I (control) dogs whose platelets aggregated in response to AA stimulation had significantly higher 24-hour platelet deposition (%IE) on the luminal surface of implanted grafts (p < 0.02), lower 4-week graft patency (p < 0.002), and higher plasma TxB₂ levels (p < 0.01) than those dogs whose platelets did not aggregate. In contrast to the results of AA stimulation, neither ADP nor collagen responsiveness was discriminatory for the thrombotic potential of canine arteries as measured by 24-hour platelet deposition (%IE), 4-week graft patency, or plasma TxB₂ levels. Pharmacologic platelet inhibition by use of aspirin (groups III and IV) effectively transformed AA responders into AA nonresponders as manifested by lowering the 24-hour platelet deposition (%IE) and increasing the 4-week patency to a value similar to AA nonresponders. This study confirms that the in vitro platelet response to AA stimulation is the best method for determining the endogenous thrombotic potential of canine arteries and correlates well with plasma TxB₂ levels and 24-hour in vivo platelet deposition studies. (J VASC SURG 1988;7:554-61.)     

Increased platelet-monocyte aggregates and cardiovascular disease in end-stage renal failure patients.

BACKGROUND: Atherosclerotic cardiovascular disease is a major cause of morbidity and mortality in patients with end-stage renal disease. This excess morbidity cannot be entirely explained by well-recognized conventional and novel risk factors alone, and occurs irrespective of dialysis modality. Recent evidence suggests that the activation of platelets and their interaction with circulating cells are important independent risk factors for atherosclerosis in non-uraemic patients. We therefore studied platelet activation and circulating platelet-leucocyte aggregates in stable patients without evidence of cardiovascular disease on continuous ambulatory peritoneal dialysis (CAPD) and haemodialysis and investigated an association with cardiovascular events. METHODS: Immunofluorescent flow cytometry was used to measure the percentage of P-selectin- (CD62P) positive platelets, the percentage of platelet-neutrophil and platelet-monocyte aggregates, and the expression of the P-selectin ligand, P-selectin glycoprotein ligand-1 (PSGL-1, CD162) on leucocytes in haemodialysis and CAPD patients and normal controls. The platelet count and the mean platelet component (MPC, a measure of platelet activation) were determined on the ADVIATM 120 Haematology System (Bayer, NY). RESULTS: Platelet activation as assessed by MPC or CD62P expression was significantly increased in haemodialysis but not CAPD patients compared with controls. Circulating platelet-monocyte aggregates were significantly increased in parallel with a significant reduction in PSGL-1 expression on monocytes in both patient groups compared with normal controls. The presence of higher platelet-monocyte aggregates in dialysis patients was associated with increased cardiovascular events. CONCLUSION: We describe increased platelet-monocyte aggregates with reduced leucocyte PSGL-1 expression in patients with end-stage renal disease irrespective of dialysis modality, associated with an increased risk of cardiovascular disease. These findings may suggest a novel mechanism by which accelerated atherosclerosis occurs in uraemic patients.     

Intraluminal albumin stent assisted laser welding for ureteral anastomosis

BACKGROUND AND OBJECTIVES: The success of laser tissue welding or soldering depends on optimal laser settings, solder material, and tissue type and geometry. To develop a practical laser welding technique for ureteral repair, an intraluminal albumin stent was designed to enhance the welding effects on ureteral end to end anastomosis. STUDY DESIGN/MATERIALS AND METHODS: In vitro porcine ureters were anastomosed using an albumin stent alone, the albumin stent plus a solder, and the solder alone. All welding was performed with an 810-nm diode laser with either a continuous wave (1 W, CW) or two pulse modes (2 W, 3.3 Hz; 1 W, 5 Hz). Laser parameters, tensile strength (TS) and burst pressure (BP) of the ureteral anastomosis, and tissue thermal injury were measured. RESULTS: In the 2-W pulse mode, BP in the albumin stent plus solder group (mean 185 mmHg) and the stent only group (mean 133 mmHg) were significantly higher than the solder only group (mean 77 mmHg, P < 0.05). Laser ureteral anastomosis with the stent plus solder group at 1-W CW and 2-W pulse laser modes yielded the highest TS (mean 97, 82 g) and BP (mean 183, 185 mmHg). Among the three modes, the 1 W pulse delivered the lowest energy and yielded the lowest TS and BP in ureteral anastomosis. There was no significant difference in the thermal damage to the tissue among the modes and groups. CONCLUSIONS: Using the albumin stent increased the reliability of ureter end-to-end laser anastomosis. Further studies will be warranted in vivo and other tubular organs as well.     

Effect of normothermic versus hypothermic cardiopulmonary bypass on cytokine production and platelet function

BACKGROUND: Proinflammatory cytokines and platelets play a key role in the systemic inflammatory response associated with cardiopulmonary bypass (CPB). The aim of this study was to evaluate the effects of both hypothermic and normothermic CPB on platelet activation, cytokine production, as well as their possible correlations. METHODS: Twenty patients who underwent CABG were randomly assigned into two groups receiving hypothermic and normothermic CPB. Blood samples were obtained through a venous catheter at 6 time points. The following parameters were measured: in vitro platelet aggregation, in vivo platelet activation, complete and differential blood cell counts, plasma soluble P-selectin levels, plasma IL-6, IL-1beta and TNFalpha levels. RESULTS: The results demonstrated that platelet abnormalities could be observed to a greater extent during hypothermic rather than normothermic CPB. The occurrence of in vivo platelet activation was suggested by the presence of a significantly increased percentage of platelets expressing CD62P on their surface, as well as by a decreased in vitro platelet aggregation induced by different agonists. Complete and differential blood cell counts showed no substantial decrease in platelet number without differences between groups. The results obtained also showed the presence of a significant release of sP-selectin during CPB, as well as a more pronounced increase of plasma sP-selectin levels in patients undergoing hypothermic compared to normothermic CPB. A comparison of cytokine levels demonstrated a significant elevation of plasma IL-6 levels during either hypothermic or normothenmic CPB, paralleling the neutrophil rise, while no differences were observed for TNF-alpha levels. Conversely, plasma IL-1beta levels were significantly elevated during hypothermic, but not during normothermic CPB. CONCLUSIONS: Hypothermic CPB is responsible for a greater platelet activation and endothelial dysfunction than normothermic CPB, leading to more profound changes in the hemostatic and inflammatory systems, which, in turn, might be responsible for the higher incidence of postoperative complications reported during hypothermic CPB.     

Platelet activation and aggregation during cardiopulmonary bypass.

Increases in plasma concentrations of platelet granule products such as platelet factor 4 and beta-thromboglobulin during cardiopulmonary bypass suggest that platelets are activated during extracorporeal circulation. Subsequent circulation of these activated platelets may be responsible for the ubiquitous platelet dysfunction associated with cardiopulmonary bypass. Using flow cytometry and a monoclonal antibody directed against an alpha-granule membrane protein, granule membrane protein 140 (GMP-140), which is expressed on the platelet surface membrane after activation, we directly measured the percentage of circulating activated platelets in 41 patients before, during, and after cardiopulmonary bypass. In addition, we compared the GMP-140 expression with platelet aggregation in response to adenosine diphosphate (ADP). Cardiopulmonary bypass produced a significant increase in the percentage of GMP-140-positive platelets persisting in the circulation; the percentage peaked at a mean of 29% (range 10-58%) before separation from extracorporeal circulation. A significant percentage of these activated platelets continued to circulate in the early postoperative period. Simultaneous measurement of platelet aggregation in response to ADP demonstrated an aggregation defect that had a time course distinct from platelet activation and whose magnitude did not correlate with the degree of platelet activation in individual patients. We conclude that cardiopulmonary bypass causes a complex constellation of platelet defects, which include alpha-granule release, prolonged circulation of activated, "spent" platelets, and impaired platelet aggregation.     

Sevoflurane anesthesia attenuates adenosine diphosphate-induced P-selectin expression and platelet-leukocyte conjugate formation

The expression of P-selectin on the surface of platelets and platelet-leukocyte conjugate formation are considered to be an indicator of platelet activation and are important in thrombotic and inflammatory disease. Previous studies have reported the inhibitory effects of sevoflurane on platelet aggregation. We investigated whether sevoflurane alters the expression of P-selectin on platelets and the formation of platelet-leukocyte conjugates. Twenty-five patients undergoing minor extremity surgery received sevoflurane-based general anesthesia, with mask induction and laryngeal mask airway anesthesia maintenance. Whole blood was obtained before and 40 min after sevoflurane anesthesia. Unstimulated and adenosine diphosphate-stimulated samples of whole blood and platelet rich plasma were stained with fluorochrome-conjugated antibodies. The expression of P-selectin on platelets and the formation of platelet-leukocyte conjugates were measured using flow cytometry. Sevoflurane inhibited platelet P-selectin expression. It also reduced the formation of platelet-leukocyte conjugates, both in unstimulated and adenosine diphosphate-stimulated blood samples at 3%-4% end-expiratory sevoflurane concentrations used to maintain anesthesia.     

Semi-solid albumin solder improved mechanical properties for laser tissue welding

BACKGROUND AND OBJECTIVE: A semi-solid albumin solder formulated with hydroxypropylmethylcellulose (HPMC) was designed to improve the characteristics of liquid and solid solders. STUDY DESIGN/MATERIALS AND METHODS: Acute tensile strengths were determined on canine small bowel in vitro by using liquid 50% bovine serum albumin (BSA), semi-solid 48% BSA with HPMC, and solid 60% BSA solder. Long-term healing of liquid and semi-solid solders, compared with a suture control, was evaluated in a porcine skin model, with tensile strength as well as histologic findings obtained on postoperative day 7. RESULTS: Acutely, semi-solid solder demonstrated a significantly (P < 0.05) higher tensile strength when compared with liquid or solid solder. At 7 days, HSA semi-solid and BSA semi-solid had significantly (P < 0.05) higher tensile strength than suture control; however, no differences were seen for liquid or solid solder groups. No differences in histology were appreciable between any of the solder groups in a porcine skin model. CONCLUSION: Acutely and at 7 days, semi-solid solder was stronger than 50% liquid albumin with better handling characteristics.     

Platelet activation is increased in peripheral arterial disease

OBJECTIVE: Platelet activation was assessed in patients with peripheral arterial disease compared with healthy control subjects. METHODS: This prospective comparative study included 100 subjects: 40 consecutive patients with intermittent claudication, 20 consecutive patients with critical ischemia and tissue loss, and 40 healthy control subjects. Whole blood flow cytometric analysis was performed to determine resting and stimulated platelet P-selectin expression and resting and stimulated platelet fibrinogen binding. Results are presented as platelet percentage and also as mean fluorescence intensity. RESULTS: P-selectin expression was significantly increased in patients with intermittent claudication (median, 0.85%; range, 0.31%-4.77%; P =.023) and critical ischemia (median, 1.11%; range, 0.2%-3.26%; P =.028) compared with control subjects (median, 0.59%; range, 0.16%-4.58%). The percentage of platelets binding fibrinogen was also significantly higher in patients with intermittent claudication (median, 2.89%; range, 1.08%-9.59%; P <.001) compared with control subjects (median, 1.57%; range, 0.17%-10.7%). There was no significant difference in percentage of platelet fibrinogen binding between control subjects and patients with critical ischemia. Fibrinogen binding by stimulated platelets was significantly diminished in patients with critical limb ischemia compared with control subjects (67.2% vs 77.9%; P =.006). CONCLUSIONS: Platelet activation is increased in patients with peripheral arterial disease, suggesting an underlying prothrombotic state. Platelets from patients with critical limb ischemia are less responsive to in vitro stimulation.     

Biodegradable polymer film reinforcement of an indocyanine green-doped liquid albumin solder for laser-assisted incision closure

BACKGROUND AND OBJECTIVE: The purpose of this study was to determine whether solid material reinforcement of a liquid albumin solder coagulum could improve the cohesive strength of the solder and, thus, the ultimate breaking strength of the incision repair in vitro. STUDY DESIGN/MATERIALS AND METHODS: A 50%(w/v) bovine serum albumin solder with 0.5 or 2.5 mg/ml indocyanine green (ICG) dye was used to repair an incision in bovine aorta. The solder was coagulated with an 806-nm continuous wave diode laser. A 50-micrometer-thick poly(DL-lactic-co-glycolic acid) film was used to reinforce the solder (the controls had solder but no reinforcement). Acute breaking strengths were measured, and the data were analyzed by Student's t-test. RESULTS: Observations of the failure modes indicate cohesive strength reinforcement of the test specimens vs. the controls. The 2.5 mg/ml ICG reinforced solder was stronger than the controls without reinforcement (P < 0.05) for all laser powers tested. There was no difference between the test specimens and the controls with 0.5 mg/ml ICG solder for low laser powers, but at higher laser powers, the reinforced solder was stronger than the controls (P < 0.05). CONCLUSION: Reinforcement of liquid albumin solders in laser-assisted incision repair seems to have advantages in terms of acute breaking strength over conventional methods that do not reinforce the cohesive strength of the solder.     

Leukocytes can enhance platelet-mediated aggregation and thromboxane release via interaction of P-selectin glycoprotein ligand 1 with P-selectin

BACKGROUND: Platelet--leukocyte conjugates have been observed in patients with unstable coronary syndromes and after cardiopulmonary bypass. In vitro, the binding of platelet P-selectin to leukocyte P-selectin glycoprotein ligand-1 (PSGL1) mediates conjugate formation; however, the hemostatic implications of these cell--cell interactions are unknown. The aims of this study were to determine the ability of leukocytes to modulate platelet agonist--induced aggregation and secretion in the blood milieu, and to investigate the role of P-selectin and PSGL-1 in mediating these responses. METHODS: Blood was drawn from healthy volunteers for in vitro analysis of platelet agonist--induced aggregation, secretion (adenosine triphosphate, beta-thromboglobulin, and thromboxane), and platelet-leukocyte conjugate formation. Experiments were performed on live cells in whole blood or plasma to simulate physiologic conditions. Whole-blood impedance and optical aggregometry, flow cytometry, and enzyme-linked immunosorbent assays were performed in the presence and absence of blocking antibodies to P-selectin and PSGL1. The platelet-specific agonists, thrombin receptor activating peptide and adenosine diphosphate, were used to elicit platelet activation responses. RESULTS: Inhibition of platelet--leukocyte adherence by P- selectin and PSGL1 antibodies decreased agonist--induced aggregation in whole blood. The presence of leukocytes in platelet-rich plasma increased aggregation, and this increase was attenuated by P-selectin blocking antibodies. Data from flow cytometry confirmed that platelet-leukocyte conjugate formation contributed to aggregation responses. Blocking antibodies reduced platelet agonist--induced thromboxane release but had no impact on adenosine triphosphate and beta-thomboglobulin secretion. CONCLUSIONS: Leukocytes can enhance platelet agonist--induced aggregation and thromboxane release in whole blood and platelet-rich plasma under shear conditions in vitro. Interaction of platelet P-selectin with leukocyte PSGL1 contributes substantially to these effects.     

Sevoflurane inhibits unstimulated and agonist-induced platelet antigen expression and platelet function in whole blood in vitro.

BACKGROUND: Previous studies have reported conflicting results about the effect of sevoflurane on platelet aggregation. To clarify this point, we investigated the effects of sevoflurane on platelet antigen expression and function in vitro. METHODS: Human whole blood was incubated for 1 h with 0.5 and 1 minimum alveolar concentration sevoflurane, 21% O(2), and 5% CO(2). A control sample was kept at the same conditions without sevoflurane. After stimulation with adenosine diphosphate or thrombin receptor agonist peptide 6, samples were stained with fluorochrome conjugated antibodies, and the expression of platelet glycoproteins GPIIb/IIIa, GPIb, and P-selectin, as well as activated GPIIb/IIIa, were measured with two-color flow cytometry. In addition, platelet function was assessed by means of thromboelastography and using the platelet function analyzer 100. RESULTS: Already in subanesthetic concentrations, sevoflurane inhibits unstimulated and agonist-induced GPIIb/IIIa surface expression and activated GPIIb/IIIa expression on platelets in whole blood. The agonist-induced redistribution of GPIb into the open canalicular system was also impaired by sevoflurane, whereas no effect on P-selectin expression in activated platelets could be found. Sevoflurane significantly reduced the maximum thromboelastographic amplitude. Furthermore, platelet function analyzer 100 closure times were significantly prolonged. CONCLUSION: The results show that sevoflurane significantly impairs platelet antigen expression in vitro. It is especially the inhibition of GPIIb/IIIa expression and activation that impairs bleeding time as reflected in thromboelastographic measurements and platelet function analyzer 100 closure times. The exact inhibitory mechanism remains unclear.     

Phosphodiesterase III inhibition affects platelet-monocyte aggregate formation depending on the axis of stimulation

OBJECTIVE: The purpose of this study was to investigate the effect of the phosphodiesterase (PDE) type 3 inhibitor milrinone on the adhesion of platelets to monocytes in vitro. DESIGN: Prospective study. SETTING: University experimental laboratory. PARTICIPANTS: Ten healthy volunteers. INTERVENTIONS: Whole blood was incubated with 1, 10, or 100 micromol/L of milrinone. After stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or adenosine-5-diphosphate (ADP), platelet-monocyte adhesion and CD11b, PSGL-1, GPIIb/IIIa, and P-selectin expression were measured by flow cytometry. MEASUREMENTS AND RESULTS: The formation of platelet-monocyte conjugates after PDE3 inhibition depended on the type of stimulation. In unstimulated and FMLP-stimulated blood platelet monocytes, aggregation was enhanced by increasing concentrations of milrinone. This augmentation was accompanied by a rise in P-selectin expression in platelets. In ADP-stimulated blood the number of platelet-monocyte aggregates decreased with increasing concentrations of milrinone. Concurrent with the reported antiinflammatory properties of PDE-inhibition, an inhibition of CD11b expression was found in monocytes after stimulation with FMLP. In contrast, in unstimulated samples lower concentrations of milrinone caused an increase in CD11b. CONCLUSIONS: These findings suggest that the effects of PDE3 inhibition on platelets and monocytes are modified by the type of stimulation and only partially suppress the inflammatory response of platelets and monocytes. The increase in platelet-monocyte conjugates in unstimulated and FMLP-stimulated blood suggested that PDE3 inhibition may also trigger proinflammatory reactions.