The increased risk of bleeding complications associated with the combination of antithrombotic agents with different mechanisms of action, have raised interest in the development of cardiovascular therapies with dual antiplatelet and anticoagulant (APAC) activity. Using naturally produced heparin proteoglycans as a framework for biosynthetic alternatives, the protein conjugated UFH chains offers a promising route [14, 19, 21]. The micro-hemorheological properties of APAC did not differ from UFH, and these data do not refer to specific safety issues for this novel naturally occurring HEP-PG mimetic.

Our in vivo study showed that dual APAC in escalated bolus doses maintained the CBC relatively stable, but after protamine neutralization, some decrease of platelet count occurred. The dosing scheme in boluses 0.25 mg/kg to 0.5 mg/kg (approximately 45–90 IU/kg of heparin) was clinically relevant but the highest dose of 0.75 mg/kg exceeds the maximal dose allowance. We have unpublished data on the administration of APAC in healthy volunteers, which do not impact the CBC values (data in file).

Both APAC and UFH enhanced RBC aggregation. At a low shear stress, an improvement in red blood cell deformability was observed with both treatments, but at higher shear stress, red blood cell deformability deteriorated, which may have contributed to the increased RBC aggregation. Compared to heparin, APAC caused similar but minor microrheological changes that were not physiologically significant in vivo. Although some hematological variables were altered, they retained within biologically normal laboratory range for the species [30]. Heparin is widely used in the clinics for the treatment of thromboembolism and as an anticoagulant, for example during the extracorporeal circulation. Initial hyper-aggregation of RBC is observed in majority of patients with cardiovascular disease. The treatment with heparin, i.e., UFH or APAC in these patients could potentially affect blood rheology [7].

Previous studies have shown that APAC was reno-protective in acute ischemic kidney injury, in contrast to UFH [21]. We also have similar preliminary experimental data in myocardial infarction (submitted) and stroke [31]. APAC as a heparin proteoglycan mimetic inhibits coagulation initiated by the intrinsic coagulation pathway and thrombin, yet it differs from conventional heparins in inhibiting platelet aggregation and procoagulant activity [21]. Collagen-induced platelet aggregation is unaffected or even enhanced by UFH, whereas APAC attenuates platelet aggregation and deposition on collagen surfaces in a dose-dependent manner [13, 14, 21]. Platelet-induced thrombosis is prevented by APAC under high shear rates, whereas in the presence of UFH, these vessels are occluded. In a collagen-coated AV shunt model [14, 21], local administration of APAC reduced both platelet and fibrin deposition. PET scans have demonstrated that APAC targets and binds with a longer retention time to the injury site compared to UFH [19, 32].

Patients with effective acetylsalicylic acid (ASA) inhibition have been reported to have lower plasma fibrinogen level and red blood cell aggregation values compared to the ineffective acetylsalicylic acid medication (ASA resistance) [33]. The fractal dimension of erythrocytes decreases with increasing aspirin concentration, so the rate of aggregation decreases [34]. At the same time, increasing aspirin concentration and shear force increase the deformation index of red blood cells, i.e., their elongation capacity improves [34]. ASA together with APAC enhances the platelet aggregation inhibition potential, albeit APAC is not directly impacting the thromboxane pathway [19].

In our study, we found that RBC deformability improved with low shear stress, but the erythrocyte elongation index moderately decreased under higher shear stress. Still, increasing the dose of UFH or APAC did not affect this effect.

Heparin-induced red blood cell aggregation remains a complex and somewhat enigmatic phenomenon within the realm of medical science, posing challenges in terms of its therapeutic implications. While heparin, a commonly used anticoagulant, is generally considered beneficial for its antithrombotic properties, its apparent role in stimulating RBC aggregation raises intriguing questions and potential concerns. RBC aggregation is influenced by many factors such as hematocrit, free radicals, and fibrinogen levels [35,36,37,38,39]. The hemoconcentration measured with hematocrit here in pigs was small, although mathematically significant, not biologically so. Previous studies have found that a significant increase in RBC aggregation occurs at higher hematocrit levels, which also shows interspecific differences [35].

One notable indicator of this phenomenon is the elevation in the erythrocyte sedimentation rate (ESR), a parameter frequently used to gauge inflammation. The increase in ESR and low shear blood viscosity are often associated with increased RBC aggregation, and this observation is especially important for patients receiving heparin therapy [7]. Underlying health conditions that already influence RBC aggregation, i.e. sickle cell anemia or diabetes, could potentially have effects from heparin-induced RBC aggregation [40].

Moreover, the promotion of RBC aggregation by heparin can have broader implications in the realm of microcirculation. The microcirculatory system, consisting of tiny blood vessels and capillaries, plays a crucial role in tissue oxygenation and nutrient delivery. However, the alterations were numerically significant, but its biological effects are not clear. It is still not known where the ‘point’ is where micro-rheological changes turn to microcirculatory deterioration.

Heparin’s impact on blood viscosity and erythrocyte sedimentation rate has been the subject of extensive research, consistently revealing its influence on the aggregation RBCs across various concentrations [7]. The data consistently indicate a noteworthy trend of increasing blood viscosity and ESR levels, aligning with a concomitant rise in RBC aggregation. This phenomenon, while intriguing, raises concerns regarding the potential adverse effects of heparin on blood rheology.

An illustrative example of heparin’s influence is the observation that, in all examined donor blood samples, the average ESR surged by approximately 75% when the heparin concentration reached 100 U/ml [7]. Such significant changes in ESR are indicative of an alteration in RBC behavior, particularly their tendency to aggregate. These findings collectively point towards a negative effect of heparin on RBC aggregation, a factor of importance in understanding its clinical implications. The decrease of platelet count in the samples might be related to a ‘relative’ in vitro decrease, supposedly related to the role of heparin in altering the red blood cell surface properties [40].

Limitations of the study include the relatively low case number and the few selected dosages for comparison. The experiments were performed in juvenile pigs with healthy vasculature, and we plan to investigate changes in pigs with atherosclerosis in the future. The experiment was a short-term study with repeated bolus dosing, and we were targeted to detect acute alterations of hematologic and hemorheological variables.