Red blood cell aging and risk of cardiovascular diseases.

We hypothesized that due to monthly bloodloss, the mechanical properties of blood of premenopausal women are superior to men, and place them at less risk for cardiovascular diseases than men in any age group. Rheological properties of blood of premenopausal women and age-matched men were compared. It was found that male blood possesses an increased viscosity, RBC aggregability and RBC rigidity. Additionally, male RBCs were found to have higher mechanical fragility. Since women in reproductive age have almost half as many old RBCs and almost twice as many young RBCs as men, we investigated the effect of in vivo aging of RBCs on their mechanical properties. Old RBCs were shown to have an increased mechanical fragility and aggregability, and decreased deformability as compared to young RBCs. Decreased deformability and increased aggregability of RBCs cause an increase in blood viscosity and are known as risk factors of cardiovascular diseases. Since men possess a higher number of old RBCs with suboptimum mechanical properties than premenopausal women, who due to monthly bloodloss have a higher number of young cells and a lower number of old RBCs than their male counterparts, our results suggest that an elevated hemorheological risk for males is associated with the age distribution of RBCs. This, in addition to significantly higher hematocrit, may be the reason for the increased risk of morbidity and mortality from cardiovascular diseases of men as compared to women of reproductive age.

Prostacyclin production at the human microvascular anastomosis: its effect on initial platelet deposition.

The human microvascular anastomosis represents a localized environment with strongly thrombotic tendencies. In previous studies, an increase in initial platelet deposition at a human ex vivo anastomosis was measured. It is postulated that this increase in anastomotic platelet deposition was due to a reduction in anastomotic prostacyclin production as a consequence of local endothelial cell injury or loss. Instead, in this study, an increase in anastomotic prostacyclin production over unsutured controls (control 1093 +/- 222 pg/ml of 6-keto prostaglandin F (PGF) 1-alpha, n=21; anastomosis 2494 +/- 414, n=21, mean +/- 1 SEM, p=0.005) is demonstrated. Anastomotic prostacyclin production was augmented by addition of arachidonic acid (0.1 mM) (39,000 +/- 11,300 pg/ml of 6-keto PGF 1-alpha, n=7, p<0.001) and suppressed by the preincubation of vessel segments with aspirin in a dose-dependent fashion (1mM) (83+/-22 pg/ml of 6-keto PGF 1-alpha, n=21, p<0.001); aspirin (0.1 mM) (312 +/- 56 pg/ml of 6-keto PGF 1-alpha, n=7, p<0.001). In further studies using a perfusion apparatus of human blood pumped through human placental artery segments, suppression of prostacyclin production did not augment initial platelet deposition (control anastomosis 4.9 +/- 2.2 x10(6) platelets per cm2, aspirin treatment 6.0 +/- 2.8 x 10(6) platelets per cm2, n=5, mean +/- 1 SEM, p>0.05). Suppression of platelet function with aspirin (0.1 mM) also did not decrease initial platelet deposition onto the anastomosis (5.8 +/- 2.8 x 10(6) platelets per cm2, n=r, p>0.05). In this model system, initial platelet deposition at the anastomosis may not be dependent upon cyclooxygenase pathways.

Quantitation of platelet deposition on human arteries: assessment of the disparity between results obtained with 111indium (111In)-labelling versus scanning electron microscopy.

Quantitation techniques for measuring platelet deposition (PD) to vessel surfaces are important to an understanding of thrombogenesis. In previous studies, scanning electron microscopy (SEM) has been shown to indicate a lower extent of PD than platelet 111In-scintigraphy. Part of this disparity may be explained by nonspecific binding of 111In to the vessel surface during perfusion, or loss of adherent 111In-labelled platelets by lysis or dissociation from the surface during specimen preparation for SEM. To assess whether these independent processes occur, we used a previously described human placental artery (HPA) perfusion model to quantify vessel 111In retention. Of the total 111In that bound to the vessel surface during perfusion, 77 +/- 42% (N = 9) was platelet associated 111In (111In-labelled platelets) and 23 +/- 19% (N = 9) was non-platelet associated 111In (nonspecific binding). After specimen fixation, 67 +/- 32% (N = 9) of the initial total surface 111In remained. This decrease is due to dissociation of both adherent 111In-labelled platelets, and nonplatelet associated 111In. After fixation, 57 +/- 34% (N = 9) of the initial total surface 111In remained as 111In-labelled platelets and 10 +/- 13% (N = 9) remained as nonplatelet associated 111In. Fixation caused no measurable lysis of platelets. These data suggest that PD may be overestimated by 111In-scintigraphy because of nonspecific binding of 111In and underestimated by SEM because of dissociation of adherent platelets during specimen preparation for SEM.

The inability of low-molecular-weight dextran, aspirin, and prostaglandin E1 to inhibit monolayer platelet adhesion to polyethylene.

In aggregometry studies employing platelet-rich plasma, monoclonal antibody 6D1 (6D1) binds to platelet membrane glycoprotein 1b (GP1b) and has been shown to abolish agglutination with ristocetin (1.5 mg/ml), with little effect on first-phase and slight diminution of second-phase aggregation with adenosine diphosphate (20 mumol/L). In perfusion studies with polyethylene microconduits (PE-100; interior diameter, 0.86 mm; length, 5 cm), platelet deposition (platelets/cm2) is restricted to a patchy monolayer when platelets are treated with 6D1 (10 micrograms/ml), providing a new model to study surface platelet adhesion isolated from platelet aggregation. The power of low-molecular-weigh dextran (DEX; mild inhibitor of fibrin formation and polymerization and von Willebrand factor-platelet interaction), aspirin (ASA; cyclooxygenase inhibitor), and prostaglandin E1 (PGE1; adenylate cyclase stimulator) to inhibit platelet adhesion on PE-100 was tested with this model. PE-100 segments were perfused with citrated human blood (5 ml, hematocrit, 35% +/- 5%) containing 6D1-treated, 111indium (111In-)labeled platelets (2.0 +/- 0.5 x 10(5) platelets/microliters) in a customized perfusion chamber (37 degrees C; flow, 1.2 ml/min; shear, 312 s-1). After a buffer flush under the same conditions, platelet deposition was measured by 111In-scintigraphy of the perfused conduits.(ABSTRACT TRUNCATED AT 250 WORDS)

Artificial microvascular graft thrombosis: the consequences of platelet membrane glycoprotein Ib inhibition and thrombin inhibition.

Early thrombosis of artificial microvascular grafts (AMG, grafts < or = 2 mm internal diameter) prevents their reliable clinical use. The present studies were undertaken to examine the effect of hirudin, a thrombin-specific inhibitor, and of the F(ab')2 fragment of PG-1, a monoclonal antibody (MoAb) directed against guinea pig platelet membrane glycoprotein Ib (GPIb), on AMG patency in an animal model. One-centimeter long segments of expanded polytetrafluoroethylene (ePTFE), 0.88 mm internal diameter, were serially implanted as interposition grafts in the guinea pig femoral arterial systems bilaterally. A control group was treated with 0.5 mL saline intravenously (IV) 30 minutes before limb 1 and limb 2 graft implantation. Three experimental groups were treated with 0.5 mL saline IV before limb 1 graft implantation as an animal control and with either 0.5 mL saline containing 1.25 mg/kg IV PG-1 F(ab')2, (which inhibits ristocetin-induced platelet agglutination and von Willebrand factor binding), hirudin 1 mg/kg IV, or a combination of both agents before limb 2 graft implantation. GPIb inhibition, thrombin inhibition, and the combination resulted in a significant prolongation of AMG patency (P < .005). Whereas thrombin inhibition with hirudin prolonged AMG patency similar to that observed with GPIb inhibition, the combination of GPIb and thrombin inhibition provided the overall longest prolongation of AMG patency. These results indicate that both platelet membrane GPIb and thrombin play a role in AMG thrombosis.

The effect of microvascular anastomosis configuration on initial platelet deposition.

The propensity for platelets to bind at a native vessel anastomosis is thought to be related to subendothelial exposure, the presence of suture material, and local flow disturbances. By using an artificial microvascular graft to artificial microvascular graft anastomosis model that mimics the geometry and topography of a native microvascular anastomosis but which eliminates the endothelial and subendothelial contributions, the influence of the normal anastomotic configuration alone on initial platelet deposition was measured. Anastomotic and immediate downstream platelet deposition was not augmented by the presence of the anastomotic configuration alone. This suggests that the enhanced initial platelet deposition in the region of a native vessel microanastomosis is primarily related to the presence of injured endothelium and exposed subendothelium rather than to flow disturbances.

Initial platelet deposition at the human microvascular anastomosis: effect on downstream platelet deposition to intact and injured vessels.

Initial platelet deposition (PD) in and around the region of a small-vessel anastomosis may set the stage for thrombosis and tissue loss. To study this problem, a human vessel model (human placental artery, HPA) has been designed to mimic the vascular injuries attendant on clinical microsurgery. To perform these studies, dissected lengths of human placental artery were treated to provide the following four types of injury: group I: control, dissected but otherwise uninjured (N = 5); group II: distal portion of vessel endothelium removed (N = 5); group III: central anastomosis, distal endothelium intact (N = 7); and group IV: central anastomosis, distal endothelium removed (N = 4). Vessels were perfused with 25 ml human whole blood for 17 +/- 5 s at an average shear rate of 536 s-1. Vessels in groups I to IV were segmented at 2-cm intervals, and the number of 111In-labeled platelets was measured. Data from the following groups of exposure zones were pooled and analyzed: endothelium intact, endothelium absent, anastomosis present, postanastomosis with endothelium intact, and postanastomosis with endothelium absent. Significant numbers of platelets were found to attach to intact endothelium, indicating that ischemia and microsurgical handling may augment platelet deposition to otherwise uninjured vessels. A similar degree of platelet deposition was measured after exposure of the subendothelium and perfusion, indicating that superficial subendothelial exposure in the absence of an additional prothrombotic stimulus may lead to no greater platelet deposition than occurs on slightly injured endothelium alone. Platelet deposition at anastomoses was strikingly elevated, although the anastomosis had no additive effect on platelet deposition to downstream endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Delivery of passivating proteins to sutures during passage through the vessel wall reduces subsequent platelet deposition by blocking fibrinogen adsorption.

Intraluminal vascular suture material, which attracts fewer than the expected number of platelets compared with the same biomaterial exposed to blood in vitro, differs from the untreated biomaterial in that it has been passed once through the vessel wall. The mechanism by which this apparently trivial maneuver reduces platelet deposition was investigated. Polypropylene suture (7-0 Prolene) was passed through human arteries (fetal and adult), and platelet deposition to the suture was measured in a standardized perfusion chamber. Single vessel passage of the sutures reduced platelet deposition by 68 +/- 23%, which contrasts sharply with the power of prostaglandin E1 (1 microM PGE1 is sufficient to abolish platelet shape change and aggregation), which inhibited only 11% of platelet deposition to the sutures. Aspirin treatment of the vessel (to prevent PGI2 formation) or endothelial stripping (to remove the ability to produce nitric oxide) had no effect on the degree of inhibition. Passage of the suture through a vessel analogue (expanded polytetrafluoroethylene) did not inhibit platelet deposition. 125I-fibrinogen adsorption to the suture after vessel passage was reduced to a degree similar to that of platelet deposition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins eluted from vessel-passed sutures revealed bands at 66, 47, and 16 kd. Western blotting indicated the presence of large amounts of albumin and hemoglobin, a moderate amount of haptoglobin, and only trace amounts of fibrinogen. When sutures were exposed to each of these proteins in vitro before perfusion, albumin and hemoglobin were found to reproduce the effect of vessel passage alone on platelet deposition. We conclude that albumin and hemoglobin adsorb to sutures during their passage through the vessel subendothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasospasm and platelet deposition in human arteries: effects of topical methylene blue.

Vasodilation of small blood vessels is controlled in part by the endothelium-derived relaxing factor (EDRF), which also inhibits platelet adhesion. Methylene blue (MB), which is occasionally applied directly to blood vessels during microsurgery to provide orientation and prevent torsion, is an irreversible inhibitor of the effects of endothelium-derived relaxing factor and may thereby augment both vasospasm and platelet responses. We have investigated the effects of the extravascular adventitial application of methylene blue on platelet deposition to human placental arteries (HPA) in the presence and absence of surgically induced vasospasm. A trend toward increased platelet deposition to human placental arteries was seen in each group but did not reach significance. The degree of platelet deposition to control human placental arteries suggests that the effects of methylene blue on platelet deposition may be dwarfed by the effects of surgical trauma and ischemia.

Thrombin generation in cardiac device recipients.

Thrombin generation measured after LVAS (4 patients) and TAH (1 patient) implantation was found to be elevated (3 times normal) in the first postoperative week and declined to normal levels when anticoagulation was begun. Thrombin generation was not elevated at the times of thromboembolic events (TIAs; N = 4 episodes).

Platelet deposition on polyethylene. Dependence of second layer platelet attachment on von Willebrand's disease plasma.

The role of von Willebrand's factor (VWF) was examined in platelet deposition on polyethylene by exposing 3/16 inch diameter discs of polyethylene to 111In labeled platelets re-suspended in citrated blood from normal and severe von Willebrand's disease donors. Discs were oscillated for 30 and 60 min, washed, fixed, counted for 111In, and examined by scanning electron microscopy (SEM). Total platelet deposition in the 0% VWF group was significantly greater than control discs at 30 min (7.0 x 10(6) +/- 0.5 compared with 3.29 x 10(6) +/- 0.6 platelets/cm2). The SEM examination revealed patches of spread platelets, with platelets adhering to platelets in controls. A dense, uniform monolayer of platelets was found on the 0% VWF discs. At 60 min, control discs had significant increases in the platelet deposition with SEM examination revealing uniform coverage of the disc with additional platelets.

Sclerotherapy of esophageal varices: long-term results and determinants of survival.

Between 1980 and 1986, 177 patients underwent sclerotherapy by means of the flexible fiberoptic endoscope for bleeding esophageal varices. Of these, 129 were treated with serial sclerotherapy alone. The remaining 48 patients underwent liver transplantation after sclerotherapy; these are reported separately. Patients were classified by Child's criteria, by the severity of the initial bleeding episode as reflected by the urgency of treatment, and by the nature of the underlying liver disease. Long-term survival rates were markedly influenced by Child's classification, with 83% of the patients in class A, 45% of those in class B, and 20% of those in class C surviving beyond 36 months (p less than 0.001). Urgent treatment was associated with a poorer survival than was elective treatment (p less than 0.001). Survival was not influenced by underlying alcoholic liver disease as compared to a nonalcoholic liver disease. The majority of deaths occurred within the first 100 days after the initial treatment. Child's class B and C patients had the highest early mortality rates, particularly in an acute treatment setting. The most frequent causes of death included progressive liver failure and persistent hemorrhage. Sclerotherapy for bleeding esophageal varices may successfully control hemorrhage, but the influence of this treatment on long-term survival is limited. Hepatic reserve, indicated by Child's classification, is the major determinant of survival. Significant improvements in survival after variceal bleeding are intimately linked to improvement in liver function.

Bleeding esophageal varices: treatment by sclerotherapy and liver transplantation.

Variceal hemorrhage is frequently a lethal event. Mortality among patients who have bled is high, with survival over the short term of only 25% to 50%. We retrospectively reviewed the records of 177 patients in whom variceal bleeding was treated with variceal sclerosis during a 5-year period from 1981 to 1986. All patients were treated by freehand injection of 25% sodium morrhuate with 35% dextrose, 4 ml per injection, through a fiberoptic endoscope. Of this group, 46 patients were treated with sclerosis followed by liver transplantation (group 1). These were compared to 36 nonalcoholic Child's class B and C patients treated with sclerosis alone (group 2). Survival at 4 years was poor in group 2 (17%). Liver failure and continued gastrointestinal bleeding were the most frequent causes of death. Survival among the liver-transplant group was significantly better (73%, p less than 0.001). Causes of death in this group were primarily due to sepsis, often in the setting of acute graft rejection. Group 1 patients were younger (39.8 +/- 10.8 vs 49.8 +/- 16.5 years, p less than 0.01); this difference is influenced by the deliberate selection of younger patients for liver transplantation. We conclude that sclerotherapy followed by liver transplantation significantly improves survival compared to conventional therapy in selected patients with advanced liver disease and portal hypertension. Donor organ availability will seriously limit the applicability of this approach to patients with bleeding esophageal varices.

A kinetic study of the effects of galactocerebroside 3-sulphate on human spleen glucocerebrosidase. Evidence for two activator-binding sites.

Extraction of control human spleen glucocerebrosidase with sodium cholate and butan-l-ol reversibly inactivates the enzyme in terms of its ability to hydrolyse the water-soluble substrate 4-methylumbelliferyl beta-D-glucopyranoside (MUGlc). The acidic brain lipid galactocerebroside 3-sulphate (sulphatide) reconstitutes beta-glucosidase activity in a strongly concentration-dependent manner. In this study we show that sulphatide exhibits three critical micellar concentrations (CMCs): CMC1, 3.72 microM; CMC2, 22.6 microM; CMC3, 60.7 microM. We designate the aggregates formed at these CMCs as primary, secondary and tertiary micelles respectively. From the results of kinetic studies performed at various sulphatide concentrations (0.012-248 microM), we found that sulphatide monomers (less than 3 microM) decreased the Km (for MUGlc) of control glucocerebrosidase from 11 to 4.6 mM, and lowered the Vmax. 2-fold. However, secondary and tertiary micelles were required for expression of high control glucocerebrosidase activities. Glucocerebrosidase prepared from the spleen of a patient with non-neuronopathic type 1 Gaucher's disease exhibited a very low Km (2.8 mM) even in the absence of exogenous lipid, and sulphatide monomers had no effect on the mutant enzyme's Km or Vmax. However, secondary or tertiary micelles markedly increased the Vmax. of the type 1 glucocerebrosidase to 60% of the corresponding control enzyme value. In contrast, for the glucocerebrosidase of the neuronopathic type 2 case, although sulphatide decreased the Km from 9.2 to 1.7 mM, the Vmax. never reached more than 5% that of the control enzyme, even at high concentrations of sulphatide. In addition, we found that secondary and tertiary sulphatide micelles enhanced the rate of inactivation of all three glucocerebrosidase preparations by chymotrypsin. Collectively, these results indicate the presence of two sulphatide-binding sites on glucocerebrosidase: one that enhances substrate binding, and another that enhances catalysis.

Sulfogalactocerebroside and bis-(monoacylglyceryl)-phosphate as activators of spleen glucocerebrosidase.

Sequential extraction of human spleen membranes with sodium cholate and n-butanol removes endogenous lipids and renders glucocerebrosidase activity dependent upon exogenous acidic lipids (e.g., phosphatidylserine, gangliosides) and a heat-stable activator protein (HSF). In the present report, we show that two previously untested lysosomal acidic lipids, namely sulfogalactocerebroside and bis-(monoacylglyceryl)-phosphate (BMP), also activate normal human glucocerebrosidase. In addition, sulfogalactocerebroside also markedly enhanced the activity of glucocerebrosidase isolated from a patient with type 1 (non-neuronopathic) Gaucher's disease, resulting in a specific activity which was 60-80% that of control glucocerebrosidase. Furthermore, when the sulfolipid was used as the activator, glucocerebrosidase from the type 1 patient was 30 times more active than the corresponding glucocerebrosidase from a person with type 2 (neuronopathic) Gaucher's disease. In contrast, the two BMPs, one rich in C26 saturated fatty acid and another rich in C18 unsaturated fatty acids, were relatively poor activators of both mutant glucocerebrosidases while providing excellent reconstitution of control activity.

Sucrose gradient analysis of phospholipid-activated beta-glucosidase in type 1 and type 2 Gaucher's disease.

Using sucrose density gradients, differences in delipidated lysosomal beta-glucosidase isolated from control spleen and spleen from patients with nonneurologic (type 1) and neurologic (type 2) Gaucher's disease have been examined. The three enzymes differ in sedimentation properties as well as in their responsiveness to activation by phosphatidylserine and heat-stable factor. The control beta-glucosidase sedimented as an apparent 45,000-Da species whose activity was dependent upon the inclusion of exogenous sodium taurodeoxycholate in the assay medium. Preincubation with a mixture of phosphatidylserine and heat-stable factor converted the control enzyme to a faster-sedimenting form which exhibited considerable activity in the absence of exogenous bile salt. Spleen beta-glucosidase from a patient with type 1 Gaucher's disease exhibited an apparent molecular weight of 154,000 on sucrose gradients. Like the control enzyme, the activity of this form was bile salt dependent. Upon preincubation with phosphatidylserine and heat-stable factor, beta-glucosidase from the type 1 case was also converted to a faster-sedimenting form which was more active in the absence of sodium taurodeoxycholate than in the presence of the bile salt. Spleen beta-glucosidase from the patient with type 2 Gaucher's disease sedimented as a broad peak of activity in the most dense regions of the sucrose gradients, appearing to be much larger than the beta-glucosidase from either the control or the type 1 Gaucher's disease patient. The activity of this large species was strongly dependent upon bile salt, and was not affected by preincubation of the enzyme with phosphatidylserine and heat-stable factor. Using the chaotropic salt, sodium thiocyanate (0.15 M), the spleen beta-glucosidase isolated from the type 1 Gaucher's disease case was converted to a slower-sedimenting species. The control enzyme sedimented slightly farther into the sucrose gradients upon treatment with the NaSCN. Thiocyanate treatment had no effect on the spleen beta-glucosidase isolated from the case of type 2 Gaucher's disease.