J. David Hellums

1993 Whitaker Lecture: biorheology in thrombosis research.

A review is presented on biorheological studies of platelet activation and platelet-platelet binding events that play key roles in thrombosis and hemostasis. Rheological methods have been used by a number of workers to establish the importance of fluid mechanical shear stress as a determinate of platelet reactions. Fluid mechanical shear stress can be regarded as a platelet agonist that is always present in the circulation and that is synergistic in its actions with other agonists. Early biorheological studies were phenomenological in that they focused on stress effects on measures of platelet function. Subsequent studies have elucidated mechanisms and have shown that the biochemical pathways of platelet activation are very different at elevated shear stresses than in the low shear stress environment used in many platelet activation studies. This finding that biochemical pathways of platelet activations are different at different shear stress levels suggests that it may be possible to develop platelet inhibitors of highly specific action: it may be possible to inhibit pathways important in thrombosis in a partially occluded artery without seriously compromising the normal hemostatic function of platelets. Another aspect of the work suggests that the biorheological approach may make it possible to develop better methods for prediction of thrombotic tendencies in human subjects.

Venous Levels of Shear Support Neutrophil-Platelet Adhesion and Neutrophil Aggregation in Blood via P-Selectin and β2-Integrin

BACKGROUND After activation, platelets adhere to neutrophils via P-selectin and beta2-integrin. The molecular mechanisms and adhesion events in whole blood exposed to venous levels of hydrodynamic shear in the absence of exogenous activation remain unknown. METHODS AND RESULTS Whole blood was sheared at approximately 100 s(-1). The kinetics of neutrophil-platelet adhesion and neutrophil aggregation were measured in real time by flow cytometry. P-selectin was upregulated to the platelet surface in response to shear and was the primary factor mediating neutrophil-platelet adhesion. The extent of neutrophil aggregation increased linearly with platelet adhesion to neutrophils. Blocking either P-selectin, its glycoprotein ligand PSGL-1, or both simultaneously by preincubation with a monoclonal antibody resulted in equivalent inhibition of neutrophil-platelet adhesion (approximately 30%) and neutrophil aggregation (approximately 70%). The residual amount of neutrophil adhesion was blocked with anti-CD11b/CD18. Treatment of blood with prostacyclin analogue ZK36374, which raises cAMP levels in platelets, blocked P-selectin upregulation and neutrophil aggregation to baseline. Complete abrogation of platelet-neutrophil adhesion required both ZK36374 and anti-CD18. Electron microscopic observations of fixed blood specimens revealed that platelets augmented neutrophil aggregation both by forming bridges between neutrophils and through contact-mediated activation. CONCLUSIONS The results are consistent with a model in which venous levels of shear support platelet adherence to neutrophils via P-selectin binding PSGL-1. This interaction alone is sufficient to mediate neutrophil aggregation. Abrogation of platelet adhesion and aggregation requires blocking Mac-1 in addition to PSGL-1 or P-selectin. The described mechanisms are likely of key importance in the pathogenesis and progression of thrombotic disorders that are exacerbated by leukocyte-platelet aggregation.

Simulation of intraluminal gas transport processes in the microcirculation

Intraluminal resistance to gas transport between the microcirculation and tissue was neglected for a half-century following the early work of Krogh. In recent years it has come to be understood that this neglect is seriously in error. This paper reviews the background for the long period of misdirection, and progress in placing the simulation of gas transport processes on a more accurate, quantitative basis.

Increased von Willebrand Factor Binding to Platelets in Single Episode and Recurrent Types of Thrombotic Thrombocytopenic Purpura

Extensive microvascular platelet aggregation is characteristic of thrombotic thrombocytopenic purpura (TTP). Previous studies have indicated that abnormalities of von Willebrand factor (vWf) are often present in TTP patient plasma. There has not been previously any direct evidence linking these abnormalities to the process of intravascular platelet aggregation in TTP. We used flow cytometry to analyze the binding of vWf to single platelets, and the presence of platelet aggregates, in the blood of 4 children with chronic relapsing (CR) TTP and 5 adults with single episode or recurrent TTP. vWf on the single platelets of CRTTP patients at all time points studied was significantly increased compared to controls, and was increased further as platelet counts decreased to levels below 40,000/μl. The single episode and recurrent adult TTP patients had platelet aggregates in the blood, as well as increased vWf on single platelets, before therapy commenced and thereafter until recovery was in process. In the one unresponsive single episode TTP patient, vWf on single platelets remained elevated, and platelet aggregates persisted, until her death. The platelet α‐granular protein, P‐selectin, was not increased on the single platelets of most TTP blood samples, suggesting that it is vWf from plasma (rather than from α‐granules) that attaches to platelet surfaces in association with platelet aggregation. These results suggest that vWf‐platelet interactions are involved in the platelet clumping process that characterizes TTP. Am. J. Hematol. 57:293–302, 1998.

Prediction of oxygen transport rates in blood flowing in large capillaries

A mathematical model has been developed to predict oxygen transport to and from blood flowing in tubes of the diameter of arterioles and larger (approximately 20 microns and larger). The resistance to oxygen transport in red cell suspensions is much higher than that of a comparable homogeneous hemoglobin solution. The increased resistance is associated with encapsulation of the hemoglobin in the red cells. Yet, somewhat paradoxically, for large capillaries relatively little resistance is within or in the immediate vicinity of the red cells. The great majority of the resistance is shown to be distributed in the plasma. Predictions of oxygen uptake and release are shown to be in excellent agreement with results of measurements taken on red cell suspensions flowing in capillaries of 27- and 100-microns diameter. The model seems to be the first for oxygen transport in flowing blood that is validated by detailed comparison with experimental results. It is a predictive model in that all parameters in the model are determined from independent measurements or from the literature.

The kinetics of platelet aggregation induced by fluid-shearing stress

The work herein examines in vitro the kinetics of platelet aggregation in response to the fluid-shearing stress imparted by a rotational viscometer. Aggregation is expressed through changes in particle size histograms and total particle count. Platelet aggregation commenced above 2000 sec-1 shear rate and was stable above 5000 sec-1. Activation by shear required less than 10 sec at all shear rates. At shear rates above 5000 sec-1, the particle count fell by one-half within 10 sec. By this point aggregates of 1000 platelets or more had appeared. Diluting the platelet suspensions suppressed shear-induced aggregation but not aggregation to ADP, supporting the hypothesis that shear-induced platelet aggregation proceeds through chemical release.

Direct demonstration of radiolabeled von willebrand factor binding to platelet glycoprotein Ib and IIb-IIIa in the presence of shear stress

In this study it is demonstrated for the first time that shear stress induces the binding of exogenous von Willebrand factor (vWF) multimers to platelets. The vWF preparations used were:125I-vWF purified from human cryoprecipitate (and including all vWF multimers present in normal plasma); and35S-cysteine-vWF secreted by human umbilical vein endothelial cells (HUVECs) (and containing unusually large vWF forms, as well as all plasma-type vWF multimers). Direct shear-induced binding to washed platelets (300–360×103/μl) of radiolabeled vWF was maximum at 60–120 dynes/cm2 evaluated at 30 sec and was in extent about one-quarter of the binding stimulated by ristocetin after 3 min of incubation. The shear-induced binding of only a small percentage of added radiolabeled vWF was sufficient to initiate aggregation. Radiolabeled vWF attached to both glycoprotein (GP) Ib and GPIIb-IIIa receptors in the shear field, with complete inhibition of binding occurring with simultaneous blockade of both receptors. Binding was potentiated by ADP released from sheared platelets.

A simple model for prediction of oxygen transport rates by flowing blood in large capillaries

A simple model has been developed for simulation of oxygen transport to and from blood flowing in conduits of the diameter of arterioles and larger (greater than or equal to 20 microns). The basis is the large capillary model (LCAP) of P.K. Nair, et al. 1989 which has been validated experimentally. Detailed calculations of the oxygen concentration distribution reveal that the dominant resistance to oxygen transport is distributed in the plasma. Relatively little resistance is present within or in the immediate vicinity of the red cells. On the basis of these findings, LCAP was simplified from four simultaneous nonlinear partial differential equations (PDEs) to one PDE by (1) assuming chemical equilibrium within the red blood cells, (2) neglecting intracellular and extracellular boundary layer resistances, and (3) incorporating transport in the cell-free region adjacent to the capillary wall into the boundary conditions. The simplified model is much easier to apply mathematically to new situations. A comparison between LCAP and the simpler model shows that they give virtually the same predictions, and the predictions agree well with experimental measurements. The model is predictive in that all the parameters are determined from the literature or from independent measurements. Thus it should be useful in studies of physiological significance, as well as in design and analysis of extracorporeal blood oxygenators.

Thrombin receptor activating peptide (SFLLRN) potentiates shear-induced platelet microvesiculation

Shear-induced activation of platelets plays a major role in vascular thrombosis. Shear stress tends to increase both platelet aggregation and procoagulant activity. One mechanism for increased procoagulant activity is promotion of the transbilayer movement of anionic phospholipids from the inner to the outer leaflet of the platelet membrane bilayer. This is accompanied by vesiculation of the platelet membrane, resulting in the formation of procoagulant membrane particles called microvesicles. In this study we have examined the effect of various platelet agonists on shear-induced platelet microvesiculation and the development of platelet procoagulant activity. Normal citrated whole blood was subjected to laminar shear rate up to 12,500 sec(-1) (shear stress approximately 375 dyne/cm2) in a cone-and-plate viscometer, and the formation of platelet microvesicles was measured by flow cytometry under different conditions. Elevated levels of shear stress induced significant microvesiculation. We investigated the effects of adenosine diphosphate, epinephrine, thromboxane A2 analog, collagen, and thrombin receptor activation peptide (SFLLRN) on shear-induced platelet microvesiculation. The thrombin peptide significantly increased shear-induced microvesicle formation. In contrast, under similar conditions, the other agonists had no significant effect on shear-induced microvesiculation. These studies suggest that thrombin formed in the vicinity of primary hemostatic plugs in areas of elevated shear stress may have a major role in the propagation of thrombi by potentiating shear-induced platelet microvesiculation.

Fibrinolysis Inhibits Shear Stress–Induced Platelet Aggregation

BACKGROUND Shear stress-induced platelet aggregation may initiate arterial thrombosis at sites of pathological blood flow. Shear stress-induced platelet aggregation is mediated by von Willebrand factor (vWf) binding to platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa. Tissue-type plasminogen activator (TPA) induces thrombolysis in coronary arteries through the local generation of plasmin. Plasmin also proteolyses GP Ib and plasma vWf. METHODS AND RESULTS Because these effects could mitigate shear stress-induced platelet aggregation, we investigated the effect of fibrinolytic agents on platelet aggregation in response to a pathological shear stress of 120 dynes/cm2 generated by a cone-and-platen rotational viscometer. Plasmin inhibited shear stress-induced aggregation of washed platelets, and this was associated with a decrease in GP Ib. TPA, at concentrations > or = 2000 IU/mL, significantly inhibited shear stress-induced platelet aggregation of platelet-rich plasma without a decrease in platelet GP Ib. In plasma-platelet mixing experiments, we determined that the TPA effect was localized to plasma. Purified vWf multimer degradation by TPA (in the presence of exogenous plasminogen) was associated with the loss of the capacity of vWf to support shear stress-induced platelet aggregation. CONCLUSIONS These results demonstrate that TPA inhibits platelet aggregation in response to pathological shear stress by altering the multimeric composition of vWf. This effect of TPA on shear stress-induced platelet aggregation may contribute, along with fibrinolysis, to the therapeutic effect of TPA in restoring blood flow during acute coronary artery thrombosis.