Presence Of Platelet Aggregates Research Articles

Aging Is Associated With Organ-Specific Alterations in the Level and Expression Pattern of von Willebrand Factor.

VWF (von Willebrand factor) is an endothelial-specific procoagulant protein with a major role in thrombosis. Aging is associated with increased circulating levels of VWF, which presents a risk factor for thrombus formation. Circulating plasma, cellular protein, and mRNA levels of VWF were determined and compared in young and aged mice. Major organs were subjected to immunofluorescence analyses to determine the vascular pattern of VWF expression and the presence of platelet aggregates. An in vitro model of aging, using extended culture time of endothelial cells, was used to explore the mechanism of age-associated increased VWF levels. Increased circulating plasma levels of VWF with elevated levels of larger multimers, indicative of VWF functional activity, were observed in aged mice. VWF mRNA and cellular protein levels were significantly increased in the brains, lungs, and livers but not in the kidneys and hearts of aged mice. Higher proportion of small vessels in brains, lungs, and livers of aged mice exhibited VWF expression compared with young, and this was concomitant with increased platelet aggregate formation. Prolonged culture of endothelial cells resulted in increased cell senescence that correlated with increased VWF expression; VWF expression was specifically detected in senescent cultured endothelial cells and abolished in response to p53 knockdown. A significantly higher proportion of VWF expressing endothelial cells in vivo exhibited senescence markers SA-β-Gal (senescence-associated β-galactosidase) and p53 in aged mouse brains compared with that of the young. Aging elicits a heterogenic response in endothelial cells with regard to VWF expression, leading to organ-specific increase in VWF levels and alterations in vascular tree pattern of expression. This is concomitant with increased platelet aggregate formation. The age-associated increase in VWF expression may be modulated through the process of cell senescence, and p53 transcription factor contributes to its regulation.

Evaluation of total leukocytes count by flow cytometry with Beckman Coulter® DXH900 in the presence of platelet aggregates

The Beckman Coulter® DXH900 uses the impedance method to measure the total leukocytes count. In presence of platelet aggregates, the device identifies the structural changes and associates an alarm with the leukocytes result. The aim of this study was to evaluate the influence of platelet aggregates using the principle of flow cytometry as a second assessment of the white blood cell count. Total leukocytes count was evaluated in 49 specimens with presence of platelet aggregates and 32 without anomaly. The differences between total leukocyte count by the two automatic methods (impedance and flow cytometry) and the microscopic method were compared. Without platelet aggregates, the median values were 5.6 (microscopic cell count), 5.4 (impedance) and 5.4 (flow cytometry) and no discordance was observed. In presence of platelet aggregates, the median values were 5.6, 6.4 and 5.1 respectively. The graphical analysis with the allowable total error range of ± 25.7% showed substantial analytical discrepancies (15/49) using impedance method while the flow cytometry method revealed minor disagreements (3/49). Analytical discordances versus WBC reference ranges showed 88% agreement and a substantial Kappa coefficient of 0.70 by impedance, while the flow cytometry method had 94% agreement and a perfect Kappa coefficient of 0.83. The formation of platelet aggregates increased the total leukocyte count performed DXH900 impedance method. Our study has shown that DXH 900 flow cytometry method may be an alternative to exclude the presence of pseudoleukocytosis. In case flags are generated, the microscopic method may be needed for the confirmation of WBC count.

Role for Platelets in Mediating Neutrophil Recruitment in TNF-α Induced Vaso-Occlusive Processes in Animals with Sickle Cell Anemia

Sickle cell anemia (SCA) pathophysiology results in chronic vascular hemolysis, and an exacerbated inflammatory state. Inflammatory molecule release and pancellular activation in the blood vessel triggers the recruitment of leukocytes and red blood cells (RBC) to the endothelial wall, and the subsequent vaso-occlusive processes that characterize the disease. A potential role for platelets in the initiation of vaso-occlusion is becoming increasingly clear, as these cells are known to form heterocellular aggregates with neutrophils and RBC in the SCA circulation and were recently shown to promote LPS-induced pulmonary vaso-occlusion in SCA mice (Bennewitz et al., JCI Insight 2017). TNF-α-triggered inflammatory processes in the cremaster microcirculation of SCA mice are often used to model vaso-occlusive processes in SCA (Turhan et al., PNAS 2002). As such, we sought to characterize the role of platelets in this model utilizing confocal and conventional intravital microscopy. Control (CON) and SCA male chimeric mice were generated by bone marrow transplantation (from C57BL6 or Berkeley SCA mice) to irradiated C57BL6 mice. After confirmation of their phenotypes, leukocyte recruitment was stimulated, or not, in mice by with the administration of TNF-α (0.5μg, i.p .) or vehicle (saline), and the cremaster microcirculation was observed by confocal and conventional intravital microscopy (the latter was employed for leukocyte adhesion quantification). For confocal microscopy, mice were administered i.v. with anti-CD41 PE, anti-CD45 PeCy-5 and anti-Ly6Gr Alexa 488, for the visualization of platelets, total leukocytes and neutrophils respectively, prior to microscopy. The effect of platelet clearance was investigated using an anti-GPIb-α (anti-CD42b) antibody (Emfret Analytics). Confocal intravital microscopy showed that, while platelets circulate freely in the cremaster microcirculation of unstimulated CON mice, the administration of TNF-α elicits, within 3h, significant platelet recruitment to the endothelium (Fig.1A), in association with the adhesion of some leukocytes (composed of both neutrophils and other leukocytes; 9.1 ± 0.8 leuk adhered 100µm-1). At 3 h post-TNF-α administration to SCA mice (Fig. 1B), considerable quantities of adhered platelets can be seen lining the endothelium and these platelets mediate the adhesion of numerous leukocytes (22.0 ± 1.8 leuk adhered 100µm-1; P<0.01, compared to CON). Of these leukocytes, 60.0±8.2% were neutrophils, and each adhered neutrophil was clearly observed as adhered in association with platelet agglomerations on the venule wall. In contrast, the rolling and adhesion of non-neutrophil leukocytes to the endothelium was rarely associated with the presence of platelet aggregates. To determine whether this platelet recruitment to the endothelium was essential for leukocyte recruitment and subsequent vaso-occlusive processes, SCA mice (n=3, for each group) received TNF-α, together with anti-CD42b (GPIbα) antibody (2μg/g, i.v.) or non-specific IgG, and leukocyte recruitment in the cremaster microcirculation was determined at 1h. Anti-CD42b almost completely depleted platelet numbers in SCA mice within 1 h (data not shown), and this clearance was associated with an inhibition of the adhesion of leukocytes to the venule walls in response to TNF-α when compared to IgG (3.8 ± 0.6 and 16.0 ± 1.6, leuk adhered 100µm-1, respectively, P<0.001). However, unexpectedly, anti-CD42b decreased the survival of SCA mice, but not CON mice, post-TNF-α administration (survival was <3h), possibly due to platelet sequestration in organs, or even due to the inhibition of platelet recruitment to the damaged endothelium. Thus, we present data to suggest that platelet recruitment to the endothelium mediates the secondary adhesion of neutrophils (but not non-neutrophil leukocytes) in the cremaster microcirculation of TNF-α-stimulated SCA mice, a frequently employed model of inflammatory vaso-occlusive processes. The role of the platelets in SCA vascular inflammation is becoming increasingly clear and these cells represent a primary pharmacological target for the prevention of vaso-occlusion in this disease. [Display omitted] DisclosuresConran:Bayer AG: Research Funding.

Von Willebrand disease biology

*Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; Department of Thrombosis and Hemostasis, C2-R Einthoven Laboratory for Experimental Vascular Medicine, LeidenUniversity Medical Center, Leiden, The Netherlands; Centre for Haematology, Imperial College London, HammersmithHospital Campus, London;§INSERM U770, Cedex, France; and –Department of Pathology and Molecular Medicine, QueensUniversity, Kingston, ON, Canada

Is There a Correlation Between Blood Type and Platelet Aggregations in the Peripheral Blood?

function, and ABO blood type. 5-8 In this pilot study, platelet aggregate data, taken as part of a routine naturopathic evaluation, was retrieved from the files of 690 adults (478 females and 212 males) attending the private naturopathic clinic of one of the authors (R.T.A.). Ethics approval for the study was granted by the Charles Sturt University Ethics in Human Research Committee. Platelet aggregates were measured and classified as described by Wu and Hoak 9 and Hoekstra et al 2 with samples that were said to be indicativeofincreasedriskofcardiovasculardiseasewherethebloodfilm contained one or more platelet aggregates measuring greater than 4 cm in diameter. ABO blood type, as determined using standard antigen-antibody agglutination, along with age and gender were also recorded. The average age for the females in this study was 44 14 years (mean standard deviation; range, 18-85 years), and the average ageformaleswas4814years(range,19-95years).Themajorityof the patients were either blood group O (44.5%) or A (42.3%), with group B accounting for 9.7% of the sample and group AB 3.5%. There were significantly more males with group AB than females (6.1% vs 2.3%; P .0217). Most patients (48%) were considered, according to naturopathic diagnosis principles, to have increased risk of cardiovascular disease based on the presence of large platelet aggregations; 28% had the presence of aggregates but were below the threshold for increased risk of cardiovascular disease, and 24% were negative for platelet aggregates. We did not find any statistically significant difference between these groups based on sex (P .889), age (P .990), or ABObloodgroups(P.317).Thenumberofplateletaggregates present, irrespective of size, was also analyzed with no statisticallysignificantassociationwithage(P.069),sex(P.159),or ABO blood type (P .886) observed. All smokers (n 7) were classed as having increased risk of cardiovascular disease; however, there was no correlation between cardiovascular risk category and medications expected to affect platelet aggregation (eg aspirin, warfarin; P .220). The association between ABO blood group and various health parameters is promoted by Peter D’Adamo, a naturopathic physician, and has been popularized in a range of Eat Right 4 Your Type books. Based on the work of D’Adamo, 10 some naturopathic practitioners use ABO blood grouping and the presence of platelet aggregates when assessing the health statusoftheirpatients,anditisbelievedtoprovideanindication of risk of cardiovascular disease. Despite many patients in this study being positive for platelet aggregates, and based on aggregate size, being deemed to be at increased risk of cardiovascular disease, we have shown that there is no statistically significant correlation between blood type and size of platelet aggregation, nor was there any association with age or sex. As this study uses retrospective data, there were some limitations with respect to the type of information included in patient files, and the influence of these factors (eg family history of cardiovascular disease, smoking, high levels of stress) on platelet aggregation in this population is unknown. According to the method used in this study, all smokers were classified as having increased risk of cardiovascular disease; however, the number of smokers was small and the validity of this finding is uncertain. This suggests that there is no scientific basis to the naturopathic diagnostic practice of lining blood type with cardiovascular risk.

Minimal platelet deposition and activation in models of injured vessel wall ensure optimal neutrophil adhesion under flow conditions.

Platelets at injured vessel wall form an adhesive surface for leukocyte adhesion. The precise relation between platelet adhesion and activation and leukocyte adhesion, however, is not known. We therefore used various models of injured vessel wall to form different patterns of platelet adhesion. The interaction of polymorphonuclear neutrophils (PMNs) was subsequently studied under flow conditions. In the absence of platelets, not only endothelial cell, smooth muscle cell, and fibroblast matrices but also purified matrix proteins (fibrinogen, collagen, and fibronectin) barely support PMN adhesion. The presence of platelets, however, strongly enhances PMN adhesion. PMN adhesion shows a proportional increase with platelet coverage up to 15%. Although PMNs roll over the scarcely scattered platelets, they speed up again when encountering surfaces without platelets. This "hopping" interaction of PMNs vanishes with platelet coverage >15%. Unobstructed rolling of PMNs is than observed and soon leads to a maximal adhesion of 1000 to 1200 cells/mm2. The mean rolling velocity of PMNs continues to decrease with higher platelet coverage. Platelet aggregate formation is an accepted indicator of platelet activation. The presence of platelet aggregates instead of contact or spread platelets, however, does not increase PMN adhesion. Also, additional stimulation of surface-associated platelets by thrombin fails to influence PMN adhesion. Moreover, indomethacin as an inhibitor of platelet activation and aggregation does not change the subsequent PMN interaction. In conclusion, approximately 15% of platelet coverage is sufficient for optimal PMN adhesion. Increasing platelet coverage increases the availability of platelet-associated receptors that lower PMN rolling velocity. Additional activation of adherent platelets makes no difference in the expression of relevant adhesion receptors. Therefore, minimal vascular damage in vivo and only scarce platelet adhesion will already evoke significant colocalization of leukocytes.

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/microl. 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 alpha-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 alpha-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.

Difference of [Ca2+]i Movements in Platelets Stimulated by Thrombin and TRAP: the Involvement of αIIbβ3-Mediated TXA2 Synthesis

This study investigated the difference of [Ca2+]i movement in platelets in response to thrombin and TRAP. The involvement of alpha(IIb)beta3 in this signaling was also studied. Stimulation of platelets with thrombin at 0.03 U/ml caused platelet aggregation and a two-peak increase in [Ca2+]i. The second peak of [Ca2+]i, but not the first peak was abolished by the inhibition of platelet aggregation with alpha(IIb)beta3 antagonists or by scavenging endogenous ADP with apyrase. A cyclooxygenase inhibitor, aspirin, and a TXA2 receptor antagonist, BM 13505, also abolished the second peak of [Ca2+]i but not the first peak, although these regents did not inhibit aggregation. Under the same assay conditions, measurement of TXB2 demonstrated that alpha(IIb)beta3 antagonists and aspirin almost completely inhibited the production of TXB2. In contrast to thrombin-stimulation, TRAP caused only a single peak of [Ca2+]i even in the presence of platelet aggregation, and a high level of [Ca2+]i increase was needed for the induction of platelet aggregation. The inhibition of aggregation with alpha(IIb)beta3 antagonists had no effect on [Ca2+]i change and TXB2 production induced by TRAP. Inhibition studies using anti-GPIb antibodies suggested that GPIb may be involved in the thrombin response, but not in the TRAP. Our findings suggest that low dose thrombin causes a different [Ca2+]i response and TXA2 producing signal from TRAP. Endogenous ADP release and fibrinogen binding to alpha(IIb)beta3 are responsible for the synthesis of TXA2 which results in the induction of the second peak of [Ca2+]i in low thrombin- but not TRAP-stimulated platelets.

The Effect of Different Antithrombotic Regimens on Platelet Aggregation After Myocardial Infarction

Platelet aggregate ratio (PAR) was measured according to the method of Wu & Hoak in 143 patients after acute myocardial infarction (AMI) and in 54 controls. A PAR < 1 expresses the presence of platelet aggregates. The patients were randomized to aspirin 160 mg/d, or warfarin, or aspirin 75 mg/d + warfarin. In patients on aspirin, PAR was measured 24 h after aspirin intake, and in 76 patients also 2 h after aspirin. The median PAR in patients on warfarin was 0.85, on warfarin + aspirin 0.91 and on aspirin alone 0.94, all significantly lower than the median PAR of 0.97 in the controls. In 14 patients on aspirin the PARs were below a cut-off point of 0.82 (secondary aspirin non-responders). PAR increased significantly 2 h after aspirin intake. In two patients, however, PAR remained low (primary aspirin non-responders). It is concluded that some patients do not seem to respond to aspirin, the clinical implication of which has yet to be determined.

Effect of aspirin on spontaneous contrast in the brachial veins of normal subjects

Blood cell aggregates are thought to be the cause of spontaneous echo contrast (SEC), although there is disagreement as to whether red cell or platelet aggregates produce this effect. One way to differentiate between these 2 possibilities is to evaluate the effect of aspirin on SEC because aspirin would not be expected to affect red cell aggregates. To eliminate the need to perform repetitive transesophageal echocardiographic studies, and the possible effect of the underlying disease process on SEC, the effect of aspirin on SEC in the brachial vein was studied in normal volunteers using a single-blind, before-and-after study design. Other factors known to affect blood echogenicity including hematocrit, sedimentation rate, and the presence of platelet aggregates by microscopy were also studied. The amount of SEC was quantitated by image analysis and expressed as the aggregate score. The results in 10 volunteers showed that all had SEC in brachial veins before aspirin, but there was no significant day-to-day variation in the amount of SEC during the control period (mean +/- SEM 104,248 +/- 23,088, 153,722 +/- 35,664, and 124,568 +/- 22,827 for days 1, 3, and 5, respectively). A significant decrease in the aggregate score occurred after 7 days of aspirin, 650 mg twice a day (51,690 vs 127,513, p = 0.002); this was accompanied by a striking decrement in the size of the largest platelet aggregate found in venous blood. Aspirin caused no significant change in the hematocrit or sedimentation rate. These results indicate that there is a component of SEC that is aspirin-sensitive and is likely to represent platelet aggregates.

Preparation and Storage in Plasma-Lyte a of Platelets Collected with the Cell Separator CS3000 plus Equipped with the PLT30-Separation and TNX6 Collection Chambers

The last five years have been characterized by the presentation of new cell separators the main task of which is the collection of high yield-high quality platelets. The CS3000 is an old apparatus which has undergone rejuvenation to sustain the assault of its new competitors. The CS3000 Plus Omnix system is the latest version to be offered along with a combination of TNX-6 separation/PLT30 collection chambers for optimal platelet collection. In this paper we present our results with this apparatus and configuration after its adaptation to the collection and storage of platelets in a non-plasma medium, the Baxter Plasma-Lyte A. After separation the platelet product (PC) was left in the collection chamber and resuspended with 200 ml of Plasma-Lyte A instead of being resuspended in autologous plasma as usual. Plasma was collected in a separate bag (400-450 ml) for transfusion or fractionation purposes. PC quality was assessed by evaluating the platelet yield (4.17 +/- 1.8 x 10(11), and the WBC contamination (4.8 +/- 2.6 x 10(5)). The presence of platelet aggregates (platelet count after aggregate fixation with formalin/platelet count after disaggregation in EDTA), the aggregation induced by ADP, collagen and ristocetin, the hypotonic shock response and the stability of membrane glycoproteins (CD 62 - 62 - 63 - 36- 42b - 51) were measured in the preapheresis samples and in the PC immediately after, 24 and 72 hours after collection. These results were totally satisfactory as was the post-transfusion survival measured as corrected count increment in 10 transfusions to non refractory patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet aggregation induced by 1-desamino-8-D-arginine vasopressin (DDAVP) in Type IIB von Willebrand's disease.

Type IIB von Willebrand's disease is a distinct form of this disorder, in which there are abnormal factor VIII/von Willebrand factor multimers in plasma (but normal multimers in platelets) and heightened interaction between the von Willebrand factor and platelets in the presence of ristocetin. We have found that infusion of desmopressin acetate (1-desamino-8-D-arginine vasopressin [DDAVP]), an agent used in the treatment of von Willebrand's disease, causes platelet aggregation and thrombocytopenia in patients with Type IIB disease. In vitro, platelets in normal plasma and those obtained from patients with Type IIB disease before DDAVP infusion aggregated upon the addition of platelet-poor plasma from Type IIB patients treated with DDAVP. Platelet aggregation was associated with adsorption of multimers of factor VIII/von Willebrand factor onto the platelets and was inhibited by EDTA. We conclude that in Type IIB von Willebrand's disease, DDAVP releases an abnormal factor with platelet-aggregating properties. DDAVP should not be used to treat patients with Type IIB disease, since the presence of platelet aggregates in the circulation may be harmful.

Fluorescence induced in platelet aggregates as a guide to luminal contours in the presence of platelet aggregation

After intravenous injection of sodium fluorescein, appropriately filtered uv light will cause platelets to aggregate in the microcirculation. These aggregates brightly fluoresce. When aggregates build up and encroach on the vessel lumen they produce an irregular contour. When such vessels are observed by ordinary in vivo microscopy, the adherent aggregates (“white bodies”) may be difficult to distinguish from the vessel wall, which may itself be difficult to define. The appearance of the red cell column then commonly becomes the basis for determining vessel contour or diameter and an erroneous impression of irregular vasoconstriction may be obtained. The bright fluorescence of the platelet aggregates under uv illumination readily corrects this impression. Aggregates induced by other means may also fluoresce under uv light if fluorescein is injected following aggregation. However, production of aggregates by fluorescein and by uv light is a method which uniquely permits simultaneous production of aggregation and precise evaluation of apparent vasoconstriction or luminal narrowing.

Endotoxins and haemostasis.

SummaryBacterial endotoxins possess the capacity to produce disseminated intravascular coagulation in laboratory animals and in man. The mechanism by which endotoxin initiates intravascular coagulation, however, has not been conclusively established. The studies reviewed in this report outline the multiple aspects of the interaction between endotoxin and the clotting system.The hypothesis that LPS initiates intravascular coagulation via the intrinsic clotting system is based predominantly on the vitro observation that it activates purified human and rabbit Hage-man factor directly.Several studies have stressed the importance of the platelets as a triggering factor in DIC. In animals, endotoxin administration is followed by thrombocytopenia, presence of platelet aggregates in the blood vessels of several organs, appearance of appreciable amounts of serotonin in plasma and increased platelet factor 3 availability. These in vivo effects can readily be explained by the in vitro observation that endotoxin aggr...