Impact of experimental haemodilution on platelet function, thrombin generation and clot firmness: effects of different coagulation factor concentrates

Abstract

Background: Haemodilution during resuscitation after massive haemorrhage may worsen the coagulopathy and perpetuate bleeding.

Materials and methods: Blood samples from healthy donors were diluted (30 and-60%) using crystalloids (saline, Ringer's lactate, Plasmalyte(TM)) or colloids (6% hydroxyethylstarch [HES130/0.4], 5% human albumin, and gelatin). The effects of haemodilution on platelet adhesion (Impact R), thrombin generation (TG), and thromboelastometry (TEM) parameters were analysed as were the effects of fibrinogen, prothrombin complex concentrates (PCC), activated recombinant factor VII (FVIIa), and cryoprecipates on haemodilution.

Results: Platelet interactions was already significantly reduced at 30% haemodilution. Platelet reactivity was not improved by addition of any of the concentrates tested. A decrease in TG and marked alterations of TEM parameters were noted at 60% haemodilution. HES130/0.4 was the expander with the most deleterious action. TG was significantly enhanced by PCC whereas rFVIIa only caused a mild acceleration of TG initiation. Fibrinogen restored the alterations of TEM parameters caused by haemodilution including those caused by HES 130/0.4. Cryoprecipitates significantly improved the alterations caused by haemodilution on TG and TEM parameters; the effects on TG disappeared after ultracentrifugation of the cryoprecipitates.

Discussion: The haemostatic alterations caused by haemodilution are multifactorial and affect both blood cells and coagulation. In our in vitro approach, HES 130/0.4 had the most deleterious effect on haemostasis parameters. Coagulation factor concentrates did not improve platelet interactions in the Impact R, but did have favourable effects on coagulation parameters measured by TG and TEM. Fibrinogen notably improved TEM parameters without increasing thrombin generation, suggesting that this concentrate may help to preserve blood clotting abilities during haemodilution without enhancing the prothrombotic risk.

Figures

Figure 1

Micrographs showing platelet interactions as observed with the cone and plate technology (Impact R). Representative microscopic fields in: (A) Experiments performed with whole blood; (B) a 30% haemodiluted sample using Ringer’s lactate; (C) a 30% haemodiluted sample using Ringer’s Lactate after in vitro addition of fibrinogen (2 mg/mL).

Figure 2

Influence of different concentrates on platelet interactions using the cone and platelet technology. Bar diagrams indicate the percentage of surface covered by platelets with whole blood (Con), after 30% haemodilution using Ringer’s lactate (RL), and after in vitro addition of fibrinogen (Fbn) at 2 mg/mL, prothrombin complex concentrates (PCC) at 35 IU/kg, activated recombinant factor VII (rFVIIa) at 6 μg/mL, and cryoprecipitates (cryo) at a dose of 2 cryo/10 kg of weight. Panel A shows the total percentage of surface covered (SC) by platelets and panel B shows the average size (AS) of the aggregates formed, expressed in μm2. Results are expressed as mean ± SEM, n=8 (*P <0.05 vs whole blood).

Figure 3

Effects of different plasma concentrates on the viscoelastic properties of clots evaluated by ROTEM. Each panel corresponds to a different fluid used for haemodilution of the sample: Ringer’s lactate (A), representative of crystalloids, and HES 130/0.4 (B), representative of colloids. Each panels shows the clotting time (CT), clot formation time (CFT) both expressed in seconds; and clot amplitude after 10 min (A10) expressed in mm. The dashed line provides the reference for undiluted control samples (Con), samples 60% haemodiluted with Ringer’s lactate (RL) or HES 130/0.4 (HES), before and after addition of: 2 mg/mL of fibrinogen (Fbn), 35 IU/kg of prothrombin complex concentrates (PCC), 6 μg/mL of activated recombinant factor VII (rFVIIa), or cryoprecipitates (Cryo) at an equivalent dose of 2 cryo/10 kg of weight. Results are expressed as mean ± S.E.M (n=8); (†) P vs control samples, (*) P <0.05 vs haemodilution (‡), and P <0.01 vs haemodilution.

Figure 4

Effect of the different factor concentrates on thrombin generation. Bar diagrams summarise modifications in (A) lag time (min) to trigger thrombin generation (lag phase) and (B): the peak of maximal thrombin concentration (nM) in samples of platelet-rich plasma (PRP) from blood haemodiluted to 60% with Ringer’s lactate. The different bars represent values in control undiluted samples (Con), haemodiluted samples before (RL), and after addition of 2 mg/mL of fibrinogen (Fbn), 35 IU/kg of prothrombin complex concentrates (PCC), 6 μg/mL of activated recombinant factor VII (rFVIIa), or cryoprecipitates (Cryo) at a dose equivalent to 2 cryo/10 kg of weight. Results are expressed as mean ± S.E.M (n=8); (*) P vs haemodiluted samples, (†) P <0.01 vs control samples, and (‡) P <0.01 vs haemodilution.

Figure 5

Effect of ultracentrifugation of cryoprecipitate on thrombin generation using samples of platelet-rich plasma (PRP) from blood haemodiluted to 60% with Ringer’s lactate. Graphs display representative thrombin generation kinetics (n=4) in control undiluted samples (◆), haemodiluted PRP (○), haemodiluted PRP plus cryoprecipitate at a dose of 2 cryo/10 kg of weight (△), and haemodiluted PRP plus supernatant of ultracentrifuged cryoprecipitates at equivalent doses (■). Enhanced thrombin generation induced by cryoprecipitates disappeared after ultracentrifugation.