Frank Brosstad

Shear-Induced Platelet Activation and Platelet Microparticle Formation at Blood Flow Conditions as in Arteries With a Severe Stenosis

In the present study, we investigated whether high arterial shear stresses at various exposure times or a sudden increase in shear stress introduced by a stenosis affect platelet activation and platelet microparticle formation in native human blood. We used a parallel-plate perfusion chamber device through which nonanticoagulated human blood was drawn (10 mL/min) by a pump directly from an antecubital vein through the flow channel of a perfusion chamber at wall shear rates of 420, 2600, and 10500 s-1. In another set of experiments, an eccentric stenosis was introduced into the flow channel. Wall shear rates of 2600 or 10500 s-1 at the stenosis apex were maintained at the same flow rate. The wall shear rate upstream and downstream of these stenoses was 420 s-1. A shear rate of 420 s-1 is within the range of those encountered in healthy small coronary arteries, whereas those of 2600 and 10500 s-1 are representative for vessels with various degrees of stenotic lesions. The blood was exposed to these shear rates for periods varying from 0.075 to 3.045 seconds. Platelet activation was assessed as activated glycoprotein (GP) IIb/IIIa by FITC-labeled monoclonal antibody (MAb) PAC-1 and aminophospholipid translocation by FITC-labeled annexin V. Microparticle formation was quantified by FITC-labeled MAb Y2/51 directed against GP IIIa. Significant platelet activation and formation of microparticles were observed at 10500 s-1 only (P < .008). This shear-induced platelet activation and microparticle formation were enhanced by introduction of a thrombus-promoting surface consisting of type III human collagen fibrils. Introduction of the most severe stenosis at 10500 s-1 further increased platelet activation (P < .017). The collagen-induced thrombus formation increased the platelet thrombus volume at 10500 s-1 from 16.5 to 33.8 microns3/microns2 (P < .003) on the stenosis apex when the most severe stenosis was used. A correlation (P < .0001) between platelet thrombus volume and platelet microparticle formation was observed in the presence of the eccentric stenoses. Apparently, high shear stress (315 dynes/cm2 at 10500 s-1), as encountered in severe atherosclerotic arteries, activated platelets and triggered platelet microparticle formation. In contrast, no significant platelet activation or formation of platelet microparticles was observed at physiological shear (420 s-1) or at the shear condition simulating shear in arteries with a less severe stenosis (2600 s-1). The data imply that platelets are activated and form microparticles in native blood at very high shear stresses. These events are potentiated by prolonged exposure to the high shear or by a sudden change of increasing shear due to the stenosis. The latter situation apparently enhances platelet thrombus formation at the stenosis.

Completely heparinized cardiopulmonary bypass and reduced systemic heparin: Clinical and hemostatic effects

BACKGROUND When heparinized circuits are used for cardiopulmonary bypass, the amounts of heparin and protamine administered systemically can be reduced. However, it is not entirely known what effects this reduction in systemic anticoagulation has on clinical performance and on the coagulation and fibrinolytic systems. METHODS Two hundred three patients undergoing first-time elective myocardial revascularization were prospectively randomized either to a group in which a completely heparin-coated circuit was used for perfusion (group H; n = 101 patients) and in which a reduced heparin dose was given (activated clotting time, > 250 seconds) or to a control group (group C; n = 102 patients) in which an uncoated, but otherwise identical, circuit was used and in which full systemic heparinization was induced (activated clotting time, > 480 seconds). Indicators of thrombin generation, platelet activation, and fibrinolytic activity were studied in a subset of 34 patients. RESULTS The total amount of postoperative mediastinal drainage was significantly reduced in group H (median, 575 mL) compared with that in group C (median, 635 mL; p = 0.002). Two patients in group C but none in group H received homologous red blood cell transfusions (p = not significant). The loss of hemoglobin in group H was a median of 21 g/L, and this was significantly lower than the 25 g/L noted in the control group (p = 0.006). During cardiopulmonary bypass, the plasma levels of thrombin-antithrombin complex and prothrombin fragment 1.2 increased in both groups. At the end of cardiopulmonary bypass the plasma levels of these markers of thrombin formation were significantly higher in group H, although the increase was modest compared with the major increase observed 2 hours after operation in both groups. There were no significant intergroup differences in the platelet counts, the concentration of beta-thromboglobulin, or the plasma levels of fibrinogen and D-dimer. No differences in perioperative morbidity, the postoperative kidney function, or the intubation time were observed, and there were no hospital deaths. CONCLUSIONS The combination of complete heparin-coated cardiopulmonary bypass circuits and low systemic heparinization is safe for patients undergoing elective coronary artery bypass procedures and reduces the perioperative blood loss. There was no evidence of increased thrombogenicity, fibrinolytic activity, or consumption of coagulation factors. No clinical or technical side effects were observed.

Absence of enhanced systemic inflammatory response at 18 weeks of gestation in women with subsequent pre-eclampsia

Objective To compare indicators of systemic inflammatory response in the second trimester in women who developed pre‐eclampsia with normal pregnancies.

Effects on Coagulation and Fibrinolysis With Reduced Versus Full Systemic Heparinization and Heparin-Coated Cardiopulmonary Bypass

BACKGROUND Extracorporeal circulation with circuits coated with surface-bound heparin has allowed reduced levels of systemic heparinization. Clinical benefits have included reduced postoperative bleeding and less homologous blood usage. However, the effects on the hemostatic and fibrinolytic systems have remained in part unknown. METHODS AND RESULTS Indications of thrombin generation, platelet activation, and fibrinolytic activity were investigated in patients undergoing coronary artery bypass surgery. Two groups were perfused with cardiopulmonary bypass (CPB) circuits completely coated with surface-bound heparin: one group with low systemic heparin dose (activated clotting time [ACT] > 250 seconds; n = 17) and a second group having a full heparin dose (ACT > 480 seconds; n = 18). A third control group was perfused with ordinary uncoated circuits and full heparin dose (n = 17). The plasma level of thrombin-antithrombin complex and prothrombin fragment 1.2 increased in all groups during bypass, and somewhat more in both the heparin-coated groups toward the end of CPB, compared with the control group (P < .01). However, the increase during CPB was minimal compared with the major elevation observed 2 hours after surgery in all groups. Platelet release of beta-thromboglobulin increased in all groups (P < .01) during CPB and significantly more in the high-dose group compared with the other two groups (P = .03). Fibrinolytic activities were similar in all groups, and there were no indications of major consumption of coagulation factors. CONCLUSIONS Reduced systemic heparinization (ACT > 250 seconds) in patients having extracorporeal circulation with completely heparin-coated circuits did not lead to increased thrombogenicity. Thrombin formation remained within low ranges during CPB compared with patients receiving a full heparin dose and with the major elevations observed after surgery.

Shear-induced platelet activation and platelet microparticle formation in native human blood

Shear-induced platelet activation and platelet microparticle formation are triggered in native human blood by high arterial shear or by a sudden increase in shear as introduced by a stenosis with potential consequences for collagen-induced platelet thrombus formation. Blood was drawn from healthy volunteers and directly perfused ex vivo over various well-defined eccentric stenoses. Shear-induced platelet activation was determined by using flow cytometry to assess: 1) GPIIb-IIIa activation by fluorescein isothiocyanate (FITC)-labeled Mab PAC-1; and 2) translocation of membrane aminophospholipids (procoagulant activity) by FITC-labeled Annexin V. Microparticle formation was measured by flow cytometry and FITC-labeled Mab Y2/51 directed against GPIIIa. Significant platelet activation and platelet microparticle formation were elicited when the wall shear rate reached 10,500 sec-1 for a period of 0.075 sec. Prolonged exposure to or a rapid increase in shear further enhanced activation and microparticle formation. Shear-induced platelet activation was associated with significantly increased collagen-induced platelet thrombus formation that was insensitive to aspirin ingestion. Exposure of native blood to very high shear thus activates platelets to express GPIIb-IIIa, renders the platelet membrane procoagulant and stimulates microparticle formation. These responses are associated with enhanced collagen-induced thrombus formation by prostaglandin-independent mechanisms.

Increased YKL-40 expression in patients with carotid atherosclerosis

OBJECTIVE We hypothesized a role for the inflammatory protein YKL-40 in atherogenesis and plaque destabilization based on its role in macrophage activation, tissue remodeling, and angiogenesis. METHODS Serum YKL-40 levels were measured by enzyme immunoassay in 89 patients with carotid atherosclerosis and 20 healthy controls. Carotid expression of YKL-40 was examined by real time RT-PCR in 57 of the patients. Regulation and effect of YKL-40 were examined in THP-1 monocytes. RESULTS Our main findings were: (1) serum YKL-40 levels were significantly elevated in patients with carotid atherosclerosis, with particularly high levels in those with symptomatic disease; (2) patients with recent ischemic symptoms (within 2 months) had higher YKL-40 mRNA levels in carotid plaque than other patients; (3) in vitro, the beta-adrenergic receptor agonist isoproterenol, toll-like receptor (TLR) 2 and TLR4 agonists, and in particular releasate from activated platelets significantly increased the expression of YKL-40 in THP-1 monocytes and (4) in vitro, YKL-40 increased matrix metalloproteinase-9 expression and activity in THP-1 monocytes, involving activation of p38 mitogen-activated protein kinase. CONCLUSIONS Our findings suggest that YKL-40 might be a marker of plaque instability, potentially reflecting macrophage activation and matrix degradation within the atherosclerotic lesion.

Peripheral endothelial dysfunction in heart transplant recipients: possible role of proinflammatory cytokines.

Endothelium‐dependent vasodilation in the peripheral circulation may be impaired in heart transplant recipients (HTx rec). Conflicting results have been obtained and the mechanisms involved have not been examined. In the present study, we examined whether long‐time survivors of heart transplantation (Tx) show signs of endothelial dysfunction in the peripheral microcirculation, and further investigated the possible role of endothelium‐related markers and proinflammatory cytokines in this process. The vasodilatory responses to acetylcholine (Ach) (endothelium‐dependent) and sodium nitroprusside (SNP) (endothelium‐independent) were evaluated by skin laser–Doppler perfusion measurements in 63 clinically stable HTx rec 6 yr (range 1–13 yr) after Tx, and compared with 20 healthy controls. Ten HTx rec were also followed prospectively with three repeated measurements during the first year after Tx. Plasma von Willebrand factor, big‐endothelin (b‐ET), and proinflammatory cytokines were measured by enzyme immunoassays. Vascular responses to both Ach and SNP were significantly attenuated in the HTx rec compared with controls. In longitudinal testing, there was a significant reduction in endothelium‐dependent vasodilation, but not independent vasodilation from 1 to 12 months after Tx. Plasma levels of vWF and b‐ET, as well as levels of proinflammatory cytokines, tumor necrosis factor (TNF)‐α, interleukin (IL)‐6 and IL‐1β, were all markedly increased in HTx rec. HTx rec responses to Ach were negatively correlated to TNF‐α levels in plasma (r=−0.39, p<0.01). Moreover, there was also a significant positive correlation between plasma b‐ET and TNF‐α (r=0.34, p<0.01). In the long‐term follow‐up of HTx rec, endothelial dysfunction is demonstrated by both regulation of blood flow in the skin microcirculation and by raised markers of endothelial activation in plasma. This endothelial dysfunction may be related to enhanced levels of proinflammatory cytokines in these patients.

Autotransfusion in coronary artery bypass grafting: Disparity in laboratory tests and clinical performance

OBJECTIVE Autotransfusion during and after cardiac surgery is widely performed, but its effects on coagulation, fibrinolysis, and inflammatory response have not been known in detail. METHODS Hemostatic and inflammatory markers were extensively studied in 40 coronary artery bypass patients undergoing a consistent intraoperative and postoperative autotransfusion protocol. An identical autotransfusion protocol was applied to 4916 consecutive coronary patients and the overall clinical results were evaluated in this large patient population. RESULTS The autologous blood pooled before bypass remained nearly inactivated after storage. A slight elevation of thrombin-antithrombin complex and prothrombin fragment 1.2, as well as plasmin/alpha(2)-antiplasmin complex was found in the content of the extracorporeal circuit after surgery, indicating thrombin formation and fibrinolytic activity. Also some increase of beta-thromboglobulin was present. In the mediastinal shed blood, complete coagulation, as evidenced by the absence of fibrinogen, had taken place and all parameters described above were extremely elevated. However, no thrombin activity was detected. As for the inflammatory response, moderately increased levels of complement activation products, terminal complement complex, and interleukin-6 traced in the extracorporeal circuit reached very high levels in mediastinal shed blood. Autotransfusion of the residual extracorporeal circuit blood and the mediastinal drainage was followed by elevation of most of these markers in circulating plasma. On the other hand, no correlating harmful effects were recorded in the study patients or in the consecutive 4916 patients. Coagulation disturbances were rare and allogeneic transfusions were required in fewer than 4% of all patients. CONCLUSIONS The hemostatic and immunologic systems were moderately activated in the autologous blood remaining in the extracorporeal circuit, whereas the mediastinal shed blood was highly activated in all aspects. However, autotransfusion had no correlating clinical side-effects and the subsequent exposure to allogeneic blood products was minimal.

Syndecan-1 plasma levels during coronary artery bypass surgery with and without cardiopulmonary bypass

The glycocalyx covering the endothelium is shed during ischemia and reperfusion. The shedding is accompanied by increased levels of the glycocalyx component syndecan-1 in the circulation. Our aim was to compare plasma levels of syndecan-1 in patients undergoing coronary artery bypass grafting (CABG), with or without the use of cardiopulmonary bypass (CPB). Syndecan-1 plasma concentrations were measured in patients undergoing CABG on-pump (n = 22) or off-pump (n = 22). The syndecan-1 concentration increased significantly from 29.5 ± 4.6 ng/mL at baseline to 98.7 ± 9.8 ng/mL (p < 0.01) after the start of CPB or 30 minutes after the induction of anesthesia in the off-pump group. There were no significant differences in peak syndecan-1 plasma concentrations between on-pump and off-pump patients. Plasma levels of syndecan-1 increased significantly during CABG, with or without the use of CPB. There were no significant differences in syndecan-1 concentrations in the two groups.

Leucocyte filtration during cardiopulmonary reperfusion in coronary artery bypass surgery

Postoperative organ dysfunction after cardiac operations has been related to the damaging effects of cardiopulmonary bypass (CPB). These complications are considered to be mediated partly by complement activation and subsequent activation of leucocytes due to the contact between blood and the large nonendothelial surfaces in the bypass circuit. Removal of leucocytes by filtration during the reperfusion period may potentially reduce the postoperative morbidity after CPB. Forty patients undergoing elective, primary coronary artery bypass grafting were randomized to initial identical bypass circuits until the aortic crossclamp was released. Then, the ordinary arterial line filter was closed and either a leucocyte depletion filter (n = 20), or a control filter (n = 20) was incorporated in the circuits during the reperfusion period of CPB. Blood samples were drawn at fixed intervals and analysed for white blood cell and platelet counts, plasma concentration of myeloperoxidase, C3-complement activation products, the terminal complement complex, and interleukins (IL)-6 and -8. The numbers of circulating white blood cells in the leucocyte-depleted group decreased during the reperfusion period from 5.5 (4.8-6.8) to 5.3 (4.4-6.2) × 109/l, and increased in the control group from 6.5 (5.1-8.0) to 7.4 (5.7-9.0) × 109/l. Two hours postoperatively the total white blood cell count in the leucocyte-depleted group was 14.7 (12.1-17.2) × 109/l, and in the control group 17.6 (14.5-20.7) × 109/l. The differences between the groups were statistical significant (p= 0.05). There were no statistically significant differences between the groups with regard to other test parameters or clinical data. We conclude that the use of leucocyte filters during the reperfusion period in elective coronary artery bypass surgery significantly reduced the number of circulating leucocytes, whereas no effects were seen for granulocyte activation measured as myeloperoxidase release, platelet counts, complement activation, or IL-6 and -8 release. The clinical benefit of leucocyte filters in routine or high risk patients remains to be demonstrated and is suggested to be dependent on both the efficacy and the biocompatibility of the filters.