C. Robert Valeri

Hypothermia-induced Reversible Platelet Dysfunction

Baboons that were subjected to systemic hypothermia at 32 C had an arm skin temperature of 27.3 C and bleeding time of 5.8 minutes. With local warming of the arm skin to 34 C, the bleeding time was 2.4 minutes. In normothermic baboons with arm skin temperature of 34.6 C, the bleeding time was 3.1 minutes. Local cooling of the arm skin to 27.6 C produced a bleeding time of 6.9 minutes. Increasing the skin temperature of the arm in hypothermic baboons to 38.9 C and in normothermic baboons to 40.1 C reduced bleeding times to 2.1 and 2.3 minutes, respectively. In both hypothermic and normothermic baboons there was a negative and significant correlation between the bleeding time and the arm skin temperature and the thromboxane B2 level in the shed blood obtained at the template bleeding time site. There was a significant positive correlation between the thromboxane B2 level in the shed blood and the arm skin temperature. Both in-vivo and in-vitro studies have shown that the production of thromboxane B2 by platelets is temperature-dependent, and that a cooling of skin temperature produces a reversible platelet dysfunction. Data also suggest that when a hypothermic patient bleeds without surgical cause, skin and wound temperature should be restored to normal before the administration of blood products that are not only expensive but may also transmit disease.

Increased Platelet Reactivity and Circulating Monocyte-Platelet Aggregates in Patients with Stable Coronary Artery Disease

OBJECTIVES We sought to examine whether patients with stable coronary artery disease (CAD) have increased platelet reactivity and an enhanced propensity to form monocyte-platelet aggregates. BACKGROUND Platelet-dependent thrombosis and leukocyte infiltration into the vessel wall are characteristic cellular events seen in atherosclerosis. METHODS Anticoagulated peripheral venous blood from 19 patients with stable CAD and 19 normal control subjects was incubated with or without various platelet agonists and analyzed by whole blood flow cytometry. RESULTS Circulating degranulated platelets were increased in patients with CAD compared with control subjects (mean [+/- SEM] percent P-selectin-positive platelets: 2.1 +/- 0.2 vs. 1.5 +/- 0.2, p < 0.01) and were more reactive to stimulation with 1 micromol/liter of adenosine diphosphate (ADP) (28.7 +/- 3.9 vs. 16.1 +/- 2.2, p < 0.01), 1 micromol/liter of ADP/epinephrine (51.4 +/- 4.6 vs. 37.5 +/- 3.8, p < 0.05) or 5 micromol/liter of thrombin receptor agonist peptide (TRAP) (65.7 +/- 6.8 vs. 20.2 +/- 5.1, p < 0.01). Patients with stable CAD also had increased circulating monocyte-platelet aggregates compared with control subjects (percent platelet-positive monocytes: 15.3 +/- 3.0 vs. 6.3 +/- 0.9, p < 0.01). Furthermore, patients with stable CAD formed more monocyte-platelet aggregates than did control subjects when their whole blood was stimulated with 1 micromol/liter of ADP (50.4 +/- 4.5 vs. 28.1 +/- 5.3, p < 0.01), 1 micromol/liter of ADP/epinephrine (60.7 +/- 4.3 vs. 48.0 +/- 4.8, p < 0.05) or 5 micromol/liter of TRAP (67.6 +/- 5.7 vs. 34.3 +/- 7.0, p < 0.01). CONCLUSIONS Patients with stable CAD have circulating activated platelets, circulating monocyte-platelet aggregates, increased platelet reactivity and an increased propensity to form monocyte-platelet aggregates.

Size dependent platelet subpopulations: relationship of platelet volume to ultrastructure, enzymatic activity, and function

A method for the separation of platelets on the basis of their size has been developed using counterflow centrifugation. Platelets were separated, free of plasma proteins and other cells, into seven subpopulations. The smallest‐sized platelets, designated as Fraction 1, had a mean platelet volume (MPV) of 3.94 ± 0.60 μm3 (SD). Each successive fraction had a progressively larger MPV. The MPV for the largest‐sized platelets, designated Fraction 7, was 8.19 ± 0.64 μm3. The MPV for the original platelets prior to fractionation was 6.57 ± 0.61 μm3. The mean density of Fraction 1 platelets was 1.067 ± 0.002 g/cm3, while Fraction 7 had a mean density of 1.072 ± 0.001 g/cm3. Transmission electron microscopy demonstrated that Fraction 1 had 4.3 ± 0.9 dense bodies per platelet, and Fraction 7 had 12.6 ± 2.4 dense bodies per platelet. Platelet LDH activity showed that the Fraction 1 platelets had 4.77 ± 0.92 iu per 1010 platelets; Fraction 7 platelets had 14.88 ± 1.23 iu per 1010 platelets. The LDH activity in the platelets before separation into subpopulations was 9.47 ± 1.45 iu per 1010 platelets.

Neutrophil and nonneutrophil-mediated injury in intestinal ischemia-reperfusion.

ObjectiveThe role of polymorphonuclear neutrophils (PMN) was examined in local and remote organ injury after intestinal ischemia-reperfusion. Summary Background DataPMN have been found to mediate the local injury in low flow intestinal ischemia-reperfusion. However, in complete intestinal ischemia-reperfusion, prevention of PMN adhesion by monoclonal antibodies to CD11b and CD18 reduces remote but not local intestinal injury. The role of PMN was further investigated in this setting. MethodsIn a rat model of 1-hour complete intestinal ischemia and 4-hour reperfusion, PMN were manipulated in the following four ways: (1) inhibition of oxygen-free radicals using manganese superoxide dismutase and catalase (SOD/CAT), (2) antagonism of PMN elastase using secretory leukocyte protease inhibitor (SLPI), (3) neutropenia using PMN antisera, and (4) inhibition of activation and adhesion using interleukin-1 receptor antagonist (IL-1ra) and tumor necrosis factor binding protein (TNFbp). Lung injury was quantified by the pulmonary permeability index, which is the ratio of bronchoalveolar lavage to blood concentration of radiolabeled bovine serum albumin, and PMN sequestration by myeloperoxidase (MPO) activity. Liver injury was estimated by PMN counts using quantitative histologic examination and by serum glutamic pyruvic transaminase (SGPT). Local injury was quantified by MPO activity and histologic grading. ResultsNeutropenia reduced the pulmonary permeability 80% from 11.0 ± 0.5 X 10−3 with saline treatment to 5.50 ± 0.12 X 10−3; IL-1ra, to 5.62 ± 0.44 X 10−3; and TNFbp, to 4.32 ± 0.18 X 10−3 (all p < 0.05). Pulmonary MPO rose from 0.03 ± 0.01 U/g to 0.25 ± 0.03 U/g (p < 0.05). This was reduced by neutropenia, 0.01 ± 0.003 U/g, but not by inhibition of oxygen-free radicals or PMN elastase. IL-1ra inhibited PMN sequestration, reducing MPO to 0.12 ± 0.01 (p < 0.05). Liver injury was 60% dependent on PMN. Ischemia-reperfusion increased SGPT from 20.3 ± 0.7 IU/L in the sham-treated animals to 97.0 ± 3.1 IU/L in the experimental animals. Neutropenia reduced this to 48.1 ± 3.9 IU/L; IL-1ra, to 44.7 ± 3.7 IU/L; SOD/CAT, to 64.0 ± 3.38 IU/L; and SLPI, to 57.1 ± 3.4 IU/L (p < 0.05 in all cases). Local injury was severe and unaffected by manipulation of the PMN. ConclusionsThese data suggest that PMN and their products mediate most of the lung, part of the liver, and none of the local gut injury after intestinal ischemia-reperfusion.

Anemia-induced increase in the bleeding time : implications for treatment of nonsurgical blood loss

BACKGROUND: Preoperative bleeding time (BT) does not correlate with postoperative bleeding in patients subjected to surgical procedures. A significant positive correlation has been reported between the BT 2 hours after cardiopulmonary bypass surgery and the nonsurgical blood loss during the first 4 hours after bypass surgery. This study was done to investigate the effect of Hct and platelet count on the BT measurement in normal, healthy men and women.

Heparin causes platelet dysfunction and induces fibrinolysis before cardiopulmonary bypass

BACKGROUND Platelet dysfunction and increased fibrinolysis are the most important etiologic factors in the hemostatic defect observed following the institution of cardiopulmonary bypass. This study examined the effects of heparin per se, administered before the institution of cardiopulmonary bypass, on platelet function and fibrinolysis. METHODS Sampling was performed in 55 patients undergoing cardiac operations before and 5 minutes after the routine administration of heparin, before the institution of cardiopulmonary bypass. RESULTS Heparin administration resulted in a significant prolongation of the bleeding time (from 6.3 +/- 2.1 to 12.6 +/- 4.9 minutes; p < 0.00001), a significant reduction in the level of shed blood thromboxane B2 (from 1,152 +/- 669 to 538 +/- 187 pg/0.1 mL; p = 0.00002), and an increase in the plasma levels of plasmin (from 11.8 +/- 9.7 to 125.4 +/- 34.8 U/L; p < 0.0001) and D-dimer (from 571.3 +/- 297.1 to 698.5 +/- 358.6 micrograms/mL; p = 0.05). There were no significant differences before and after heparin administration in the plasma levels of fibrinogen, plasminogen, tissue plasminogen activator, antiplasmin, antithrombin III, and von Willebrand factor. CONCLUSIONS Heparin, independent of cardiopulmonary bypass, causes both platelet dysfunction and increased fibrinolysis. The use of an alternative anticoagulant or a lower dose of heparin in conjunction with heparin-coated surfaces might improve the hemostatic balance during open heart operations.

Myocardial depression during sepsis

The cardiac response to volume loading was evaluated in fifty severely septic patients. After a rapid infusion of albumin or whole blood the cardiac index (CI) and left ventricular stroke work index (LVSWI) were recorded as the pulmonary arterial wedge pressure (PAWP) increased. Initial values of PAWP, CI, and LVSWI were similar in both the nineteen surviving and thirty-one nonsurviving patients. Surviving patients, however, demonstrated greater increases in CI and LVSWI as PAWP rose. Nearly half of both patient groups developed decreases in CI and LVSWI as the PAWP continued to increase. These downslopes occurred at relatively low PAWP and are taken as evidence of an abnormality of myocardial function in both survivors and nonsurvivors. The lower upslope of the performance curves in nonsurvivors indicates myocardial depression or a negative inotropic effect. Cardiac ischemia, acute respiratory failure, and high affinity red cells were found to diminish the cardiac response to volume loading, whereas hepatic and renal failure were associated with a good CI and LVSWI response.

Rapamycin Ameliorates Proteinuria-Associated Tubulointerstitial Inflammation and Fibrosis in Experimental Membranous Nephropathy

Proteinuria is a risk factor for progression of chronic renal failure. A model of proteinuria-associated tubulointerstitial injury was developed and was used to examine the therapeutic effect of rapamycin. Two studies were performed. In study A, proteinuric rats were given sheep anti-Fx1A to induce experimental membranous nephropathy; control rats received normal sheep serum. Four weeks later, groups were subdivided and underwent laparotomy alone (two kidneys), nephrectomy alone (one kidney), or nephrectomy with polectomy (0.6 kidney). Renal function and morphology were evaluated 4 wk later. Whereas control rats never developed proteinuria, anti-Fx1A induced severe proteinuria. Proteinuria was unaffected by renal mass reduction. Proteinuric rats developed tubulointerstitial disease that was most severe in rats with 0.6 kidneys. Renal function (GFR) was reduced by loss of renal mass and was reduced further in proteinuric rats with 0.6 kidneys. In study B, the effect of rapamycin on the expression of candidate proinflammatory and profibrotic genes and the progression of proteinuria-associated renal disease were examined. All rats received an injection of anti-Fx1A and were nephrectomized and then divided into groups to receive rapamycin or vehicle. Gene expression, renal morphology, and GFR were evaluated after 4, 8, and 12 wk. Rapamycin reduced expression of the proinflammatory and profibrotic genes (monocyte chemotactic protein-1, vascular endothelial growth factor, PDGF, TGF-beta(1), and type 1 collagen). Tubulointerstitial inflammation and progression of interstitial fibrosis that were present in vehicle-treated rats were ameliorated by rapamycin. Rapamycin also completely inhibited compensatory renal hypertrophy. In summary, rapamycin ameliorates the tubulointerstitial disease associated with chronic proteinuria and loss of renal mass.

Reduction of bleeding after heart operations through the prophylactic use of epsilon-aminocaproic acid

UNLABELLED Excessive postoperative bleeding after heart operations continues to be a source of morbidity. This prospective double-blind study evaluated epsilon-aminocaproic acid as an agent to reduce postoperative bleeding and investigated its mode of action. One hundred three patients were randomly assigned to receive either 30 gm epsilon-aminocaproic acid (51 patients) or an equivalent volume of placebo (52 patients). In a subset of these patients (14 epsilon-aminocaproic acid, 12 placebo), tests of platelet function and fibrinolysis were performed. RESULTS By multivariate analysis, three factors were associated with decreased blood loss in the first 24 hours after operation: epsilon-aminocaproic acid versus placebo (647 ml versus 839 ml, p = 0.004), surgeon 1 versus all other surgeons (582 ml versus 978 ml, p = 0.002), and no intraaortic balloon versus intraaortic balloon pump use (664 ml versus 1410 ml, p = 0.02). No significant differences in platelet function could be demonstrated between the two groups. Inhibited fibrinolysis, as reflected by less depression of the euglobulin clot lysis and no rise in D-dimer levels, was significant in the epsilon-aminocaproic acid group compared with the placebo group. CONCLUSION The intraoperative use of epsilon-aminocaproic acid reduces postoperative cardiac surgical bleeding.

Comparison of the effects of transfusions of cryopreserved and liquid-preserved platelets on hemostasis and blood loss after cardiopulmonary bypass

OBJECTIVE The aim of the study was to compare the clinical effects and hemostatic efficiency of transfusions of platelets preserved in the frozen state for as long as 2 years with transfusions of platelets preserved in the conventional manner for as long as 5 days in patients undergoing cardiopulmonary bypass. METHODS Seventy-three patients were prospectively randomly assigned to receive transfusions of cryopreserved or liquid-preserved platelets. Nonsurgical blood loss was measured during and after the operation. Bleeding time, hematologic variables, and the bleeding time site shed blood were assayed before cardiopulmonary bypass and at 30 minutes and 2, 4, and 24 hours after transfusion. In vitro platelet function tests were conducted on platelets obtained from healthy volunteers. RESULTS No adverse sequelae of the transfusions were observed. Blood loss and the need for postoperative blood product transfusions were lower in the group receiving cryopreserved platelets. Lower posttransfusion platelet increments and a tendency toward decreased platelet survival were observed in patients receiving cryopreserved platelets. Hematocrit and plasma fibrinogen were significantly higher in this group, and the duration of intubation was shorter. In vitro, cryopreserved platelets demonstrated less aggregation, lower pH, and decreased response to hypotonic stress but generated more procoagulant activity and thromboxane. CONCLUSIONS (1) Cryopreserved platelet transfusions are superior to liquid-preserved platelets in reducing blood loss and the need for blood product transfusions after cardiopulmonary bypass. (2) The reduction in blood loss in the patients receiving cryopreserved platelet transfusions after cardiopulmonary bypass probably reflects improved in vivo hemostatic function of cryopreserved platelets. (3) Some in vitro measures of platelet quality (aggregation, pH, hypotonic stress) may not reflect in vivo quality of platelet transfusions after cardiopulmonary bypass, whereas other in vitro measures (platelet procoagulant activity and thromboxane) do.