Plasma Free Blood Research Articles

Plasma-Free Blood as a Potential Alternative to Whole Blood for Transcriptomic Analysis

RNA sequencing (RNAseq) technology has become increasingly important in precision medicine and clinical diagnostics, and emerged as a powerful tool for identifying protein-coding genes, performing differential gene analysis, and inferring immune cell composition. Human peripheral blood samples are widely used for RNAseq, providing valuable insights into individual biomolecular information. Blood samples can be classified as whole blood (WB), plasma, serum, and remaining sediment samples, including plasma-free blood (PFB) and serum-free blood (SFB) samples that are generally considered less useful byproducts during the processes of plasma and serum separation, respectively. However, the feasibility of using PFB and SFB samples for transcriptome analysis remains unclear. In this study, we aimed to assess the suitability of employing PFB or SFB samples as an alternative RNA source in transcriptomic analysis. We performed a comparative analysis of WB, PFB, and SFB samples for different applications. Our results revealed that PFB samples exhibit greater similarity to WB samples than SFB samples in terms of protein-coding gene expression patterns, detection of differentially expressed genes, and immunological characterizations, suggesting that PFB can serve as a viable alternative to WB for transcriptomic analysis. Our study contributes to the optimization of blood sample utilization and the advancement of precision medicine research.

The GPIbα intracellular tail - role in transducing VWF- and collagen/GPVI-mediated signaling.

The GPIbα-VWF A1 domain interaction is essential for platelet tethering under high shear. Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GpIbaΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail that harbors the 14-3-3 and phosphoinositide-3 kinase binding sites. GPIbαΔsig/Δsig platelets bound von Willebrand factor (VWF) normally under flow. However, they formed fewer filopodia on VWF/botrocetin in the presence of a aIIbb3 blocker, demonstrating that despite normal ligand binding, VWF-dependent signaling is diminished. Activation of GPIbαΔsig/Δsig platelets with ADP and thrombin was normal, but GPIbαΔsig/Δsig platelets stimulated with collagenrelated- peptide (CRP) exhibited markedly decreased P-selectin exposure and aIIbb3 activation, suggesting a role for the GpIba intracellular tail in GPVI-mediated signaling. Consistent with this, while hemostasis was normal in GPIbαΔsig/Δsig mice, diminished tyrosine-phosphorylation, (particularly pSYK) was detected in CRP-stimulated GPIbαΔsig/Δsig platelets as well as reduced platelet spreading on CRP. Platelet responses to rhodocytin were also affected in GPIbαΔsig/Δsig platelets but to a lesser extent than those with CRP. GPIbαΔsig/Δsig platelets formed smaller aggregates than wild-type platelets on collagen-coated microchannels at low, medium and high shear. In response to both VWF and collagen binding, flow assays performed with plasma-free blood or in the presence of aIIbb3- or GPVI-blockers suggested reduced aIIbb3 activation contributes to the phenotype of the GPIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbα in transducing both VWF-GPIbα and collagen-GPVI signaling events in platelets.

How clinicians can minimize transfusion-related adverse events?

ObjectivesTransfusion-related adverse events (TRAE) can contribute to patient morbidity and mortality. In this brief narrative review, the strategies that clinicians can apply at the bedside to avoid TRAE are discussed. MethodsStrategies to avoid the following five types of TRAE were reviewed: transfusion-associated circulatory overload (TACO), transfusion-related acute lung injury (TRALI), transfusion-associated hypothermia (TAH), transfusion-related allergic reactions (TRAR) and acute haemolytic transfusion reactions (AHTR). ResultsMinimizing exposure to blood components is fundamental to TRAE avoidance. Pre-transfusion assessment can identify patients at risk of TACO, TRAR and TAH, and avoidance steps implemented. Preventive strategies for TACO include lower transfusion rate, ‘one unit at a time’ transfusion policy and possibly diuretic medication. Patients with past history of TRAR should preferably be given plasma-free blood components; anti-histamine medication prior to transfusion could be considered. TAH is common in the massive transfusion setting, particularly trauma patients. Warming of patients are key strategies to avoid TAH. Identification of patients at risk of TRALI is more opaque; however, any measures that limit pulmonary inflammation prior to transfusion may decrease the risk of TRALI. Causes of AHTR are commonly due to human error and failure to apply rigorous cross-checks of patient and issued RBC component blood groups. ConclusionsBeneficial strategies to avoid TRAE include judicious use of blood components, identification of high-risk patients, adherence to recommended clinical processes and awareness of TRAE pathophysiology. More evidence is warranted to better guide clinicians in the prevention of TRAE.

The Von Willebrand Factor D'D3 Domains Modulate the Interacton of Platelet Receptor Glycoprotein Ib with the VWF-A1 Domain Under Conditions of Shear Stress

Abstract 2212The VWF A1 domain contains the binding site for the platelet receptor glycoprotein (GP)Ib receptor, and this interaction forms the initial tethering event to capture platelets at sites of injury under high shear stress. The VWF-A1:GPIbα interaction has a high association and dissociation rate and as such platelets translocate along the VWF surface until stable attachment can occur. It has been previously shown that under static conditions the VWF D’D3 domains shield the A1 from interaction with GPIbα, however it has not been established if this occurs under conditions of shear stress. To this end we constructed VWF fragments spanning the D’-A3 domains and the A1A2A3 domains. These were expressed and purified and the ability of these constructs to interact with platelets was analysed in an in vitro flow assays system. Purified and fluorescently labelled platelets resuspended in plasma free blood were perfused over immobilised VWF at a range of shear rates (400–2000s−1), and real time movies of the interactions captured. Interestingly, platelets translocated slower across VWF-A1A2A3, with lower rolling velocities compared to VWF-D’A3 at all shear rates tested. Significantly the dissociation rate constant (koff) of the transient tethers formed between GPIbα and VWF was similar for both constructs, indicating that that the observed difference in platelet rolling velocity is not due to an alteration in bond lifetime. Frame-by-frame analysis of platelet translocation demonstrated that platelets paused more often on VWF lacking the D’D3 domains and therefore translocated a shorter distance in a set time when compared to VWF D’A3 resulting in decreased rolling velocities. We hypothesise that the accessibility of the GPIb binding site in the A1 domain is increased in the absence of the D’D3 domains, resulting in more interactions. Together these data demonstrate the D’D3 domains are able to shield the A1 domain under shear stress conditions. Disclosures:No relevant conflicts of interest to declare.

99mTc-tricarbonyl labeled agents for cell labeling: Development, biodistribution in normal mice and preliminary in vitro evaluation

We have developed four 99mTc(CO) 3-labeled lipophilic tracers as potential radiolabeling agents for cells based on a hexadecyl tail. 99mTc(CO) 3-hexadecylamino- N, N′-diacetic acid (negatively charged), 99mTc(CO) 3-hexadecylamino- N-α-picolyl- N′-acetic acid (uncharged), 99mTc(CO) 3- N, N′-dipicolylhexadecylamine (positively charged), 99mTc(CO) 3- N-hexadecylaminoethyl- N′-aminoethylamine (positively charged) were prepared in a radiolabeling yield: >90%. Preliminary cell uptake studies were performed in mixed blood cells with or without plasma and were compared with 99mTc-d,l-HMPAO and [ 18F]FDG. In plasma-free blood cells, maximum uptake (78%) was obtained for 99mTc(CO) 3- N-hexadecylaminoethyl- N′-aminoethylamine after 60 min incubation (compared to 55% and 23% for 99mTc-d,l-HMPAO and [ 18F]FDG, respectively) while in plasma-rich medium, 99mTc(CO) 3- N, N′-dipicolylhexadecylamine was best bound (54%, similar to the binding of 99mTc-d,l-HMPAO). Biodistribution in normal mice showed mainly hepatobiliary clearance of the agents and initial high lung uptake. The radiolabeled compounds showed good blood clearance with maximally 7.9% injected dose per gram at 60 min post injection. While the least lipophilic agent ( 99mTc(CO) 3- N, N′-dipicolylhexadecylamine, log P = 1.3) showed the best cell uptake, there appears to be no direct correlation between lipophilicity and tracer uptake in mixed blood cells. In view of its comparable cell uptake to well known cell labeling agent 99mTc-d,l-HMPAO, 99mTc(CO) 3- N, N′-dipicolylhexadecylamine merits further evaluation as a potential cell labeling agent.

Activation-Independent Platelet Adhesion and Aggregation under Elevated Shear Stress.

The response of platelets to vascular injury involves adhesion onto reactive substrates followed by activation and aggregation, the latter mediated by the integrin αIIbβ3 with bound adhesive proteins such as fibrinogen and von Willebrand factor (VWF). Hemostasis and pathological arterial occlusion, however, occur in distinct hydrodynamic environments. A 90% lumen reduction in a coronary artery may cause shear rates of 20,000–40,000 s−1, values that are 10-fold higher than in arterioles where platelets participate in hemostasis after trauma. How hemodynamic forces influence platelet function is not fully understood. We have now found that platelets adherent to an immobilized VWF surface in a flow field can promote aggregation without activation, provided that soluble VWF be present and the shear rate exceed 10,000 s−1. In these studies blood was treated with 10 μM prostaglandin (PG) E1, thus the definition of aggregation is extended to a process of platelet cohesion mediated by physiologic blood components even in the absence of activation. Upon blood perfusion, individual rolling platelets covered the VWF surface at the shear rate of 3,000 s−1. In contrast, at 20,000 s−1 rolling platelets formed aggregates within 100–200 μm from the point of initial contact with immobilized VWF, equivalent to a few seconds. These aggregates became larger with varying shape and velocity while translocating in the direction of flow, but were predominantly elongated during periods of prolonged arrest. We then used plasma-free blood cells suspended in buffer to evaluate whether addition of soluble VWF was sufficient to mediate activation-independent platelet aggregation. Single platelet adhesion to immobilized VWF was the same with or without soluble VWF at the shear rate of 3,000 s−1. At 24,000 s−1, in contrast, adhesion was minimal without soluble VWF, but in its presence activation-independent aggregates formed as in whole blood. To elucidate the nature of the links that support activation-independent platelet aggregation, we used reflection interference contrast microscopy (RICM). This showed that, from a discrete point of tight adhesion and over a period of several seconds, the platelet body was stretched by the fluid drag into a structure reaching lengths of 20 μm or more but less than 1 μm thick at the narrowest point. Additional platelets then adhered to these elongated ones, and some that were stretched by the fluid drag contributed to the increasing length of string-like formations, while many retained their discoid morphology. In the end, stretched platelets joined to one another formed continuous structures that reached lengths of 100–200 μm and remained attached to immobilized VWF for minutes, while hundreds of discoid platelets adhered to them and to one another with arrest times of variable duration until they eventually detached as rolling aggregates. Thus, stretching of platelets by fluid drag and binding of soluble VWF are required to form elastic links that connect platelets into activation independent aggregates.

Recombinant factor VIIa enhances platelet adhesion and activation under flow conditions at normal and reduced platelet count

Recombinant factor VIIa (rFVIIa), which was developed for treatment of inhibitor-complicated hemophilia, appears suitable as prohemostatic agent in other clinical disorders including patients with thrombocytopenia. It is generally accepted that rFVIIa functions by enhancement of thrombin generation at the site of injury. It is, however, unknown if and how this affects platelet adhesion and aggregation. To determine the effect of rFVIIa-mediated thrombin generation on platelet adhesion and aggregation under flow conditions at normal and reduced platelet counts. Washed platelets and red cells were combined to obtain plasma-free blood with different platelet counts. The reconstituted blood was perfused over a collagen- or fibrinogen-coated surface in the absence or presence of a thrombin generating system consisting of purified coagulation factors rFVIIa, factor (F)X and prothrombin. Addition of coagulation factors rFVIIa, FX and prothrombin to washed platelets and red cells enhanced platelet adhesion and aggregation to collagen and adhesion and spreading to fibrinogen at normal platelet count and at platelet numbers as low as 10 000 microL(-1). rFVIIa-mediated thrombin generation enhanced the activation state of platelets as measured by intracellular calcium fluxes, and enhanced the exposure of procoagulant phospholipids as measured by annexin A5 binding. Taken together, increased platelet adhesion and aggregation by rFVIIa-mediated thrombin formation may explain the therapeutic effects of rFVIIa in thrombocytopenic conditions and in patients with a normal platelet count by (i) enhancement of primary hemostasis and (ii) enhancement of procoagulant surface leading to elevated fibrin formation.

Mutations in the von Willebrand Factor A1 Domain That Increase the Affinity for Collagen Have Different Effects on the Binding to Glycoprotein Ib.

The interaction of plasma von Willebrand factor (VWF) with collagen at the site of vascular injury plays a critical role in the initiation of thrombus formation under high shear stress. It does this by forming a bridge between the fibrils of collagen in the subendothelium and the platelet glycoprotein Ib-IX-V complex (GPIb). The A1 domain of VWF is the binding site for GPIb whereas the collagen-function of VWF is controlled by both A1 and A3 domains. The VWF-A3 domain is important to support binding to fibrils of collagen Types I and III while the A1 domain is involved in the binding to microfibrillar collagen Type VI. It is assumed that the interaction of VWF with fibrillar collagen (via the A3 domain) may regulate the expression of the GPIb-binding site in the A1 domain. However, there is no a definite data to substantiate that hypothesis. Our goal was to demonstrate that a direct interaction between the A1 domain and fibrillar collagen Types I or III exposes the GPIb binding site. Thus, we postulated that platelet GPIb is able to interact with isolated A1 domain that is bound to collagen. We have demonstrated that the VWF-A1 protein binds specifically to human placenta collagen Types I and III with a KD ~ 200 nM by using surface plasmon resonance (SPR). Using plasma-free blood, we have provided strong evidence that isolated VWF-A1 domain bound to either collagen Type I or III is able to support platelet adhesion under high flow conditions. This platelet interaction was effectively blocked with antibodies against either GPIb or A1 domain. These results clearly show the ability of the A1 domain to concurrently interact with both GPIb and collagen fibrils and they also suggest that the collagen-A1 binding may regulate the expression of the GPIb-binding site in the A1 domain. To test this hypothesis, we analyzed three residues that in a previous mutagenesis study they increased the binding of VWF to GPIb, reasoning that they may have an effect on the collagen binding activity as well. The three residues are located in the a7 helix (rear face) of the folded A1 domain and mutagenesis studies of other I(A)-domains have demonstrated that this helix plays a role in regulating the affinity of the ligand-binding. We introduced point mutations into the 3 residues and the recombinant mutant proteins were expressed in bacteria. The three mutants (R687E, D688R, and E689R) were purified as wild type and their structural integrity was confirmed with three conformation-specific antibodies. All the mutants bound to both collagens Type I or III with an affinity much higher than the wild type (WT) (KD~ 9 -1 nM). The mutants were assessed by their ability to mediate platelet adhesion to collagen, and their ability to inhibit both ristocetin-induced platelet agglutination and shear-induced platelet aggregation. Interestingly, in the three assays the R687E mutant had an activity higher than WT while the D688R had a markedly decrease activity. The mutant E689R had an activity similar to that of WT for the three assays. Together our data indicate that a direct association between the VWF-A1 domain and collagen fibrils influences the expression of GPIb binding function in VWF. Further, these data indicate that residue R687 located in the a7 helix plays a novel and important role in modulating the collagen/A1/GPIb binding.

Stabilization of Red Blood Cell Membranes by Thalidomide In Vitro

The anti‐inflammatory effect of thalidomide has been well established. The mechanism of this anti‐inflammatory action is still not completely understood. Certain drugs exert their anti‐inflammatory action by stabilizing the membranes of polymorphonuclear neutrophils (PMN) thereby reducing the production of reactive oxygen intermediates. We evaluated the effect of thalidomide on cell membranes by using red blood cells (RBC), PMN and the monocyte‐like cell line THP‐1. Osmotic fragility of RBC showed that in vitro, thalidomide stabilized the membrane of RBC from plasma free blood; whereas, it did not affect RBCs from whole blood. Red blood cells taken from subjects before and after ingestion of thalidomide were not affected after exposure to different concentrations of hypotonic NaCl solution. Thalidomide did not affect the membrane stability of PMNs as well as THP‐1 in a significant manner. These data suggest that the anti‐inflammatory mechanism of thalidomide is not related to events associated with the oxidative burst of PMNs or monocytes.