Platelet interactions with the endothelium and the subendothelium: the role of thrombin and prostacyclin.

The adherence of 51Cr-labeled platelets to rabbit aortae everted on probes rotated in platelet-red cell suspensions has been measured. Platelet adherence to the subendothelium exposed by passage of a balloon catheter before everting the aortae was inhibited by compounds that increase platelet cyclic AMP levels (PGE1, PGI2 or dipyridamole). These agents, however, did not abolish platelet adherence to the subendothelium. Aspirin treatment of the vessel wall was used to block PGI2 production; platelet adherence to the surface of the 'undamaged' aorta and the subendothelium was studied following this treatment. Since aspirin treatment of the 'undamaged' vessel wall did not cause platelets to adhere to it, it seems unlikely that PGI2 formation by the vessel wall is the mechanism that prevents platelet adherence to normal endothelium. In addition, PGI2 formation by the vessel wall does not appear to influence platelet adherence to the subendothelium, since adherence was not increased by aspirin treatment of the damaged wall. Thrombin treatment of the 'undamaged' vessel wall increased platelet adherence to the surface, but the adherent platelets were seen to be adherent only to small areas where the endothelium was lost or damaged. Heparin reversed the effect of thrombin. Similar results were found when the subendothelium was exposed to thrombin or thrombin and heparin.     

Von Willebrand factor in the vessel wall mediates platelet adherence

A monoclonal antibody directed against the von Willebrand factor moiety (vWF) of factor VIII-von Willebrand factor (FVIII-vWF), which blocks ristocetin-induced platelet aggregation as well as the binding of FVIII- vWF to platelets in the presence of ristocetin, inhibited platelet adherence to human artery subendothelium when present in normal flowing blood. This monoclonal antibody, CLB-RAg 35, inhibited platelet adherence as a function of the shear rate. At wall shear rates below 500 s-1, platelet adherence was not affected, but at higher shear rates platelet adherence was gradually inhibited, reaching an average of 11% of the normal value at 2,500 s-1. Indirect immunofluorescence established the reactivity of CLB-RAg 35 with vWF present in artery subendothelium. Pretreatment of normal vessel walls with this antibody inhibited adherence of platelets in blood from a patient with severe homozygous von Willebrand's disease and in blood from normal individuals. The inhibition was shear-rate dependent and significant at high shear rates (2,500 s-1). By adding increasing amounts of purified FVIII-vWF to normal blood, the inhibition was gradually overcome. These data indicate that vWF present in the vessel wall contributes appreciably to platelet adherence. At high wall shear rates, platelet adherence is mediated virtually completely by both plasma FVIII-vWF and vWF in the vessel wall. At low wall shear rates (below 500 s-1), platelet adherence occurs independent of FVIII-vWF in plasma and vWF in the vessel wall.     

Retention in glass bead columns as a measure of leucocyte adhesiveness. II. Influence of platelets and erythrocytes.

The influence of platelets and erythrocytes on the retention of leucocytes in glass bead columns was studied. Leucocyte retention increased when platelet retention was stimulated by the presence of erythrocytes. In the presence of only a few erythrocytes, platelet retention was low, and platelets did not influence leucocyte retention. When only a small number of platelets was present, erythrocytes inhibited leucocyte retention. The platelet-dependent leucocyte retention increased as the volume passed through the columns was increased. However, the platelet-independent leucocyte retention did not change when the sample size was increased. In blood from patient with thrombocytopenia or defect in platelet function and in normal blood treated with inhibitor of platelet adhesion, neutrophil retention was reduced. However, erythrocyte-free leucocyte suspensions from these cases showed normal leucocyte retention. Thus, experiments with whole blood may serve as a model for the study of platelet-leucocyte interaction. However, for the study of leucocyte adhesiveness per se, isolated leucocytes seem more suitable than whole blood.     

The Influence of Red Blood Cells on the Effects of Aspirin or Sulphinpyrazone on Platelet Adherence to Damaged Rabbit Aorta

The effects of acetylsalicylic acid (ASA; aspirin) or sulphinpyrazone (SP) on the adherence of washed rabbit platelets to the subendotheilial surface of an everted aorta mounted on a probe or to the subendothelial surface of a rabbit aorta attached to a perfusion apparatus were examined. ASA had no effect on platelet adherence to a damaged aorta perfused with a suspension of washed platelets in a medium containing 10% red blood cells (RBC); SP was slightly inhibitory at high concentration. When damaged rabbit aortae were everted on a probe and rotated in a suspension of washed platelets to which RBC were added to a packed cell volume of 10%, both ASA and SP inhibited platelet adherence to the damaged vessel wall. When the PCV was 40%, ASA was not inhibitory and SP reduced platelet adherence only at very high concentrations. It is therefore unlikely that, at the concentrations achieved in man, SP exerts an effect on platelet adherence. The different effects of ASA and SP on platelet survival do not appear attributable to their effects on platelet adherence.     

Studies on gastric mucosal microcirculation. 1. The nature of regional variations induced by ethanol injury.

BACKGROUND/AIMS: The focal nature of gastric ulcers raises the possibility of underlying regional disturbances in gastric mucosal microcirculation. This study employed fluorescent in vivo microscopy with the aim of directly investigating the response of several areas of the gastric mucosa to 60% ethanol. METHODS: Changes in macromolecular leakage of fluorescein labelled albumin, vessel diameter, and acridine red labelled leucocyte adhesion and rolling were assessed over a period of two hours. A total of 0.5 ml 60% ethanol was topically applied for five minutes to the exteriorised gastric mucosa of anaesthetised rats. RATS: Three distinct patterns of response were found. Areas of lesion formation were small and occurred within five minutes. These areas showed persistent blood flow stasis throughout the course of the experiment, increased leakage (p < 0.02), and no leucocyte adhesion. Peripheral to the lesion, sustained leakage (p < 0.02) was found with adherence of leucocytes (p < 0.01) after lesion formation. Sites more remote to any lesion showed transient leakage and significant numbers of 'rolling' leucocytes (p < 0.01) were observed again after the lesion had formed. CONCLUSIONS: Despite widespread exposure of the entire gastric mucosa to 60% ethanol the resultant mucosal injury was limited. Widespread vascular damage was found reflected by macromolecular leakage, the pattern of which showed regional variation.     

Modification of leucocyte adherence inhibition (LAI) assay with 51Cr-labelled leucocytes (author's transl)

Leucocyte adherence inhibition was measured by a new modified radioisotopic technique. Peripheral blood leucocytes were isolated and labelled with Chrom51 (51Cr). These leucocytes were incubated with medium of buffer alone and with medium or buffer containing tumor antigen or gluten. The glass surface for the adherence was prepared carefully. At all samples the adherence of leucocytes occurred under the same conditions. The authors gave a report on the results of their leucocyte adherence inhibition (LAI) assay with gluten and stomach cancer antigen.     

Leucocyte-endothelial cell adhesion in a model of intestinal inflammation.

Leucocyte-endothelial cell adhesion is modulated by a variety of adhesion glycoprotein expressed on the surface of leucocytes and endothelial cells. Although in vitro studies show that these adhesion molecules mediate the decrease in leucocyte rolling velocity and the increase in leucocyte adherence and emigration associated with inflammation, there are few in vivo data to support this hypothesis. The aim of this study was to assess the role of leucocyte (CD11b/CD18) and endothelial cell (P- and E-selectin) adhesion molecules in mediating the leucocyte-endothelial cell adhesion elicited in rat mesenteric venules during a model of longlasting intestinal inflammation. Indomethacin was injected 48 and 24 hours before the experiment. The mesenteric microcirculation was observed by intravital microscopy in animals treated with monoclonal antibodies (MAb) directed against either P-selectin, E-selectin, or CD11b/CD18. Leucocyte rolling velocity, and the number of adherent and emigrated leucocytes as well as vessel diameter and erythrocyte velocity were monitored in roughly 30 micron diameter postcapillary venules. Indomethacin treatment resulted in mucosal ulceration and granulocyte infiltration, and a corresponding inflammatory response in the mesentery, which was characterised by an increase in the number of adherent (eightfold) and emigrated (sixfold) leucocytes and a reduction (80%) in leucocyte rolling velocity. The indomethacin induced leucocyte-endothelial cell adhesion in mesenteric venules was significantly reduced by treatment with MAbs against either CD11b/CD18 or E-selectin, but not by the P-selectin MAb. These results suggest that both leukocyte (CD11b/CD18) and endothelial cell (E-selectin) adhesion molecules contribute to the granulocyte accumulation in a chronic model of intestinal inflammation.     

Leucocyte function in patients with rheumatoid arthritis: quantitative in-vivo leucocyte mobilisation and in-vitro functions of blood and exudate leucocytes.

Quantitative leucocyte mobilisation in vivo and the in-vitro random migration, chemotaxis, phagocytosis, and oxidative metabolic activity were studied in 15 patients with rheumatoid arthritis (RA). Patients mobilised leucocytes to chambers covering skin windows to the same degree as control subjects, and the mobilisation correlated with the blood leucocyte numbers and serum concentration of alpha-l-antitrypsin. Peripheral blood leucocytes showed slightly reduced migration in Boyden chambers but increased phagocytosis and increased unstimulated reduction of nitroblue tetrazolium. Exudate leucocytes from patients with RA showed migratory and phagocytic activity which did not differ from that of control subjects, but unstimulated exudate leucocytes reduced nitroblue tetrazolium more actively than leucocytes from control subjects. The observations indicate that leucocyte accumulation at an experimental inflammatory lesion and the function of these exudate leucocytes are not impaired in patients with rheumatoid arthritis.     

Thrombotic thrombocytopenic purpura plasma enhances platelet–leucocyte interaction in vitro

In thrombotic thrombocytopenic purpura (TTP), intravascular platelet aggregation and formation of platelet-rich thrombi impair the microcirculation. TTP plasma has been shown to induce aggregation of normal platelets in vitro . The present study investigates the formation of activated platelet aggregates (aPAg) induced by TTP plasma, with particular attention to their binding to leucocytes (LPAg). Results were compared with the effects of plasmas from normal controls (CTL) and from patients with immune thrombocytopenic purpura (ITP) or thrombosis (THR). Following addition of test plasma to normal whole blood (WB), aPAg and LPAg were assayed by flow cytometry using mAbs against CD41 (platelet marker), CD62p (platelet activation marker) and CD45 (pan-leucocyte marker). Compared to control plasma, TTP plasma was more potent than ITP or THR plasma in increasing aPAg; only TTP plasma significantly promoted leucocyte binding to give increased LPAg. Prior removal of neutrophils (PMN) from WB by beads coated with anti-CD15 mAb largely prevented formation of aPAg and LPAg. However, TTP plasma added to normal platelet-rich plasma significantly increased aPAg, which suggested possible hindrance of aPAg formation by erythrocytes and other leucocytes in PMN-depleted blood. We concluded that TTP plasma was most potent in the induction of aPAg and unique in promoting LPAg formation in WB. Neutrophils, and not other leucocytes, appear to be essential for LPAg formation. Enhanced PMN–platelet interaction in the microcirculation may facilitate platelet adhesion to vessel walls and promote the formation of platelet-rich microthrombi in TTP.     

Plasma histamine concentrations are elevated in patients with diabetes mellitus and peripheral vascular disease

Previous work has shown that plasma and tissue concentrations of histamine are elevated in rats with experimental diabetes mellitus and that leucocytes and platelets from patients with peripheral vascular disease have a higher histamine content than those from controls. In the present study, we have measured: (a) plasma histamine concentrations; (b) leucocyte and platelet histidine decarboxylase (the enzyme responsible for the biosynthesis of histamine) in patients with diabetes mellitus (Types I and II) and peripheral vascular disease; and (c) platelet and leucocyte histamine content. Plasma histamine concentration was significantly higher in patients with diabetes and peripheral vascular disease respectively than that in age-matched controls. Leucocyte histidine decarboxylase activity in diabetic and peripheral vascular disease patients was similar to that in controls, while platelets had no histidine decarboxylase activity. The leucocyte and platelet content of histamine were greater in patients with peripheral vascular disease than those in controls, but they were not altered in diabetic patients. There was no correlation between plasma histamine concentration, leucocyte and platelet histamine content, and histidine decarboxylase activity. We conclude that plasma histamine is elevated in diabetics and in patients with peripheral vascular disease and that platelet and leucocyte histamine content is increased in the latter. This increase in platelet and leucocyte histamine content is not due to an increase in histidine decarboxylase activity of these cells. The increase in plasma and cellular histamine content may contribute to the pathogenesis of increased endothelial permeability in diabetes and to the pathogenesis of intimal damage in atherosclerosis.     

The relationship between vessel wall injury and venous thrombosis: an experimental study

The relative importance of stasis, vessel wall damage and hypercoagulability in the pathogenesis of venous thrombosis remains disputed. While the combination of local vascular stasis and systemic hypercoagulability can be shown to produce experimental thrombi within a few minutes, it has been claimed that vessel wall damage is also a necessary component of venous thrombogenesis. In this experimental study, mechanical crushing of the jugular veins produced patchy areas of denuded endothelium, with underlying vessel wall oedema, as seen by ultrastructural examination. While the exposed subendothelium became covered with activated platelets following restored blood flow, there was no fibrin formation after 5 min. When blood flow was restored for 60 min following the crush injury, white cells could be seen adhering to and migrating through the vessel wall, although there was still no visible fibrin. The addition of venous stasis for 20 min did not lead to the formation of stasis thrombi in association with the damaged areas. The present experiments demonstrate that, far from there being subtle endothelial damage contributing to acute venous thrombosis, even readily demonstrable damage is a poor stimulus to fibrin formation at local sites of vessel wall injury.     

In vitro Evaluation of a New Filter for Leucocyte Depletion of Platelet Concentrate during Component Preparation

A newly developed filter for prestorage leucocyte depletion of platelet concentrates (PC) was studied. The filter is designed for leucocyte depletion during the preparation of the pool PC from platelet rich buffy coats. In all the leucocyte depleted PC (LD-PC) leucocyte depletion was satisfactory. 19 of 20 units of LD-PC had a leucocyte content below 3times10⁵per PC and 1 contained 8times10⁵leucocytes. The standard PC contained 2.53times10⁸(0.87times10⁸–15.3times10⁸; n = 20) leucocytes per PC (median and range). The quality of the LD-PC was evaluated by measuring platelet activation, platelet morphology, and pre- and poststorage pH. There were no differences in any of the parameters studied.     

Leucocyte migration inhibition test in coeliac disease - a reappraisal.

Results of the direct leucocyte migration inhibition (LMI) test using gluten fraction III as antigen were unaffected by incorporation of puromycin into the culture medium at concentrations shown to prevent lymphokine mediated inhibition. Results of the LMI test performed with purified polymorphs were similar to and correlated with results of the standard LMI test using mixed leucocytes in both coeliacs and controls. The addition of purified T lymphocytes did not increase migration inhibition. Normal leucocytes incubated with serum from coeliac patients and washed showed marked migration inhibition when incubated with gluten fraction III. This sensitisation of normal leucocytes was prevented by preincubation with aggregated human IgG. These results suggest that leucocyte migration inhibition by gluten in coeliac disease is not due to lymphokine production by sensitised lymphocytes but is caused by cytophilic antibody.     

Factor VIII-von Willebrand factor requires calcium for facilitation of platelet adherence

The role of divalent cations in platelet adherence to deendothelialized human arteries in flowing blood was investigated in an annular perfusion chamber. Spreading of platelets on the subendothelium was impaired below 30 microM of free Ca2+ ions (Ca2+). When Ca2+ was replaced by Mg2+, adherence was unchanged in perfusates without exogenous factor VIII-von Willebrand factor (FVIII-vWF), but the ability of FVIII-vWF to support platelet adherence was lost. Binding of FVIII-vWF to the vessel wall was independent of divalent cations, but bound FVIII-vWF was only able to mediate adherence after exposure to Ca2+. Pretreatment of FVIII-vWF with the calcium chelator EGTA (10 mM) resulted in loss of the ability to facilitate platelet adherence, while the ristocetin cofactor activity remained intact. Full restoration of the ability to mediate platelet adherence could only be obtained by prolonged dialysis against Ca2+ in the millimolar range. These data indicate that divalent cations have at least two separate roles to play in supporting platelet adherence: (1) platelet spreading on the subendothelium requires Ca2+ or Mg2+; (2) FVIII-vWF should be exposed to Ca2+ to obtain its optimal biologic activity in supporting platelet adherence.     

Defective responsiveness to ascorbic acid of neutrophil random and chemotactic migration in Felty''s syndrome and systemic lupus erythematosus.

Polymorphonuclear (PMN) leucocytes from 4 patients with untreated systemic lupus erythematosus (SLE) showed defective random migration (P less than 0-05) and depressed chemotactic responses to C5a and kallikrein (P less than 0-01) compared to PMN leucocytes from normal subjects, or patients with rheumatoid arthritis (4) or Felty's syndrome (4) when examined at a standardized cell concentration with a micropore filter radioassay but not with a conventional Boyden technique. Normal in vitro enhancement of PMN leucocyte random and chemotactic migration by sodium ascorbate was absent in SLE and Felty's syndrome, but sodium ascorbate gave normal stimulation of hexose monophosphate shunt activity in the PMN leucocytes precluding a defect in ascorbate transport.     

Leucocyte migration inhibition with collagen type I and collagen type III in rheumatoid arthritis and degenerative joint diseases

The leucocytes of 22 patients with classic rheumatoid arthritis were investigated with the direct leucocyte migration inhibition technique in agarose as described by Clausen. The leucocytes of 9 patients with degenerative joint diseases and of 9 healthy persons served as controls. Collagen type I and collagen type III were used as antigens. The leucocytes of 22 patients with rheumatoid arthritis showed in 15 cases a migration inhibition with collagen type III and in 10 cases with collagen type I. The inhibition with collagen type III was stronger and more frequent than with collagen type I. The frequent leucocyte migration inhibition with collagen type III in classic rheumatoid arthritis seems to be an expression of possible cell mediated reactivity against that type of collagen which appears especially in rheumatoid synovial tissue.     

Cellular immunity to possible synovial antigens in rheumatoid arthritis.

A reaction has been demonstrated between extracts of synovial cells removed from intact rheumatoid knee joints and autologous leucocytes. The cell mediated immunity test system used was leucocyte migration inhibition. Variable reactions were found with a spectrum of allogeneic extracts when donor leucocytes came from married or transfused females or transfused males. Leucocytes from healthy (nontransfused) males showed no reaction with any of the extracts. As a period of cell culture was used prior to preparation of this extract to remove nonspecific inhibitory substances, native immunoglobulins, and complexes, the data are best explained by the presence of a foreign pathogen or altered cell component in the synovial cells of these rheumatoid patients.     

Red blood cell size is important for adherence of blood platelets to artery subendothelium

The hematocrit is one of the main factors influencing platelet adherence to the vessel wall. Raising the hematocrit causes an increase of platelet accumulation of about an order of magnitude. Our studies concern the role of red cell size. We have studied this effect using an annular perfusion chamber, according to Baumgartner, with human umbilical arteries and a steady-flow system. Normal human red blood cells (MCV 95 cu mu) increased platelet adherence sevenfold, as the hematocrit increases from 0 to 0.6. Small erythrocytes from goats (MCV 25 cu mu) caused no increment in adherence in the same hematocrit range. Rabbit erythrocytes (MCV 70 cu mu) caused an intermediate increase in adherence. Red blood cells from newborns (MCV 110-130 cu mu) caused a larger increase in platelet adherence than normal red cells at hematocrit 0.4. These results were further confirmed with large red blood cells from two patients. Experiments with small red cells (MCV 70 cu mu) of patients with iron deficiency showed that platelet adherence was similar to normal red cells, provided the red cell diameter was normal. Small red blood cells of a patient with sideroblastic anemia caused decreased adherence. These data indicate that red cell size is of major importance for platelet adherence. Red cell diameter is more important than average volume. However, for size differences in the human range, the hematocrit remains the dominant parameter.     

Leukotriene B4 mediates shear rate-dependent leukocyte adhesion in mesenteric venules.

Previous studies have demonstrated that low shear rates promote leukocyte adherence to microvascular endothelium in postcapillary venules. The objective of this study was to determine whether an accumulation of inflammatory mediators such as platelet activating factor and leukotriene B4 is responsible for shear rate-dependent leukocyte-endothelial cell adhesion. Postcapillary venules (25-39 microns in diameter) in cat mesentery were studied by intravital microscopy. Venular wall shear rate was varied over a wide range by graded occlusion of the mesenteric artery. Red blood cell velocity, vessel diameter, leukocyte rolling velocity, and the numbers of rolling and adherent leukocytes were measured at each shear rate. In one series of experiments, shear rate-dependent leukocyte adherence was monitored at different superfusion rates (1.0 and 2.5 ml/min). At the lower superfusion rate, the number of adherent leukocytes was significantly higher at any given shear rate when compared with results obtained at the higher superfusion rate. This suggests that reduced washout of inflammatory mediators contributes to shear rate-dependent leukocyte adhesion. Pretreatment with different platelet activating factor receptor antagonists (WEB 2086 or WEB 2170) had no effect on the number of adherent leukocytes normally observed at lower shear rates, suggesting that platelet activating factor does not play a major role in this process. However, shear rate-dependent leukocyte adhesion was largely prevented by pretreatment with either a leukotriene B4 receptor antagonist (SC-41930) or a leukotriene synthesis inhibitor (L663,536). The results of this study indicate that a reduced washout of leukotriene B4 is responsible for the enhanced leukocyte adherence that occurs at low venular wall shear rates.     

Migration inhibition with various cell fractions in human colorectal cancer.

The unseparated leucocytes, separated mononuclear cells and granulocytes of six control subjects and nine patients with colorectal cancer have been studied by a direct cell migration inhibition technique. A migratory index was calculated from the migration in the presence and absence of a perchloric acid extract of large bowel tumours. In 10% homologous AB serum, no significant migration inhibition occurred with any of the cells from control subjects. Five of the nine cancer patients showed significant inhibition with their unseparated leucocytes, seven of seven with their mononuclear cells, and none of nine with their granulocytes. In 10% autologous serum, some controls exhibited migration inhibition with their unseparated leucocytes and their granulocyte fraction, but not with the mononuclear cell fraction. Migration inhibition was also now apparent in the granulocyte fraction of the cancer patients. It is concluded that, with a soluble tumour antigen preparation, a mononuclear cell population increases the sensitivity of the direct migration inhibition test and that autologous serum may interfere directly with the migration of granulocytes, by an action not dependent upon the release of inhibitory factors from sensitized lymphocytes. This could explain some of the inconsistencies of the assay when using an unseparated leucocyte population.