Q&A For Biomedical Researchers

Note: The following is a discussion of the “state of the science” in the area of

thrombolysis and thrombectomy for intervention in stroke. Our intention is to highlight specific particulars in this field of knowledge for the interested biomedical researcher.

Opus-14™ is medical device under development for the treatment of ischemic stroke by revascularization of the occluded artery and is not approved for sale or

distribution in the United States or internationally.

1) Opus process works by ultraviolet light. How do you dissolve a clot with such wavelength of light?

First, the method does not require that the clot be irradiated with UV light. This indirect property is unique in the medical literature. Instead, the arterial wall just proximal to the clot is irradiated circumferentially with UV (in the clinical application, endovascularly). The quantum energy contained in the UV photons is sufficient to release the well-known vasodilator agent, nitric oxide, from its precursors (nitrites and nitrosothiols) stored in the smooth muscle cells comprising the arterial wall. By this method (known as photoscission), nitric oxide can be produced at far greater local concentration than by any enzymatic or pharmacological means. Four major effects result: (1) immediate (in seconds) dilation of the artery at the proximal end of the clot, (2) destabilization of densely organized loci of aggregated platelets in the clot, resulting in dilation of the clot itself concomitant with (3) formation of continuously branching microchannels in the clot as the platelets become unbound, permitting multiple pathways for exit of the platelets from the clot and perfusion of blood into and through the clot, and (4) continuous transport (spreading) of the dilation effect along the artery in both proximal and distal directions, possibly by a neurogenic mechanism.

2) What is nitric oxide?

Nitric oxide (chemical formula NO) is the simplest chemical known to dilate arteries. This discovery was recognized by the 1998 Nobel Prize for Medicine, awarded to Furchgott, Ignarro and Murad.

Where does it come from in the Opus process?

Nitric oxide is normally produced by enzymes (called nitric oxide syntheses) and it is involved in many fundamental processes. Its production in the endothelium (the single cell layer overlying the inner wall of an artery) governs the ability of the vessel to constrict or dilate rapidly in response to local conditions. As explained above, however, the UV laser produces NO directly from smooth muscle cell stores by literally cracking the electronic bonds that sequester it in its precursors. The main photophysical source of NO now appears to be nitrites, which compensate for their poor specific absorbance by their high tissue concentration (ca. 10 uM). From literature values, we are able to predict that a UV laser intensity of 10 watts/cm2 can convert 90% of the nitrite contained within an arterial wall into NO within 10 seconds.

3) What does dethrombosis mean?

This refers specifically to disaggregation of that portion of thrombi comprised of aggregated platelets, in which adjacent platelets are bound together by fibrinogen cross-links between surface receptor molecules (called GPIIb-IIIa proteins) on different platelets. Fibrin is not involved in this mechanism, nor is it needed, as the intraplatelet/fibrinogen cross-links are quite strong enough to withstand the force of arterial flow without it.

4) Clot busting drugs are approved for stroke. Why are the clot-busters not working?

Use of the tPAs (tissue plasminogen activators) in stroke is not common (fewer than 5% of patients - perhaps 2% - receive it) owing to widespread fear that an ischemic (clot-induced) stroke could be converted to a hemorrhagic stroke (symptomatic intracerebral hemorrhage), which is much harder to manage and more acutely severe. tPA was developed to dissolve clot-binding fibrin. Fibrin consists of a long string of cross-linked fibrinogen molecules, which presumably forms a mesh (in conjunction with activated platelets) which can entrap red blood cells. The tPAs are presumed to be clot-specific, meaning that it targets an enzyme called plasminogen which is already taken up into a clot, and the resultant reaction is supposed to dissolve the fibrin. Unfortunately, occlusive clots extracted from living stroke patients contain a significant fraction of tightly aggregated platelets, which are organized into layers which are contiguous with but separate from fibrin, also appearing in layers. In effect, the fibrinogen cross-linked platelet layers are refractory to tPA and therefore shield the fibrin from tPA. If the main clot cannot be penetrated, tPA will tend to dissolve existing clots that were formed under normal conditions to maintain hemostasis (i.e., prevent hemorrhage). These clots normally contain just enough tPA to enable their remodeling and reclamation of a smooth vascular surface.

5) What is bad about the mechanical devices for breaking up a clot or removing a clot?

Every mechanical method (including ultrasound and even tPA) intended to break up clots intentionally produces emboli, which supposedly are small enough to be scavenged by white blood cells. This has never actually been proven, however, and the resultant uncertainty has led to the development of meshworks to trap these fragments. When tPA is successful, emboli are still produced in the form of platelet aggregates. The MERCI retriever certainly intends to extract clots wholly, but clots may be fragmented during deployment. Worse, if the clot resists extraction altogether or is extremely difficult to penetrate, platelets are suspected. The pressure of blood can compact an existing thrombus and even (if the obstruction is an embolus) facilitate platelet binding to the arterial wall. Attempts to force removal in this case can lead to arterial dissection. Arterial wall damage is sustained in any case, so the remaining surface is still thrombogenic even if the lumen is “cleared.”

6) Why is the effect of Opus dethrombosis dependent on nitric oxide?

Platelets aggregated and bound together by fibrinogen cross-links mediated by GPIIb-IIIs receptors will not respond to tPA, but will disaggregate under thrombin inhibition. That is how dethrombosis was originally discovered, but the clot models used originally were rent by seepage channels, which facilitated easy entry of the drugs. Our UV method was tested on pure platelet thrombi, which were so tightly bound (as in the clinical case) that no intracellular space was available for penetration of drugs. The only type of chemical capable of penetration such as matrix is a gas, and indeed, NO is a gas. NO apparently works by inhibiting an enzyme upstream of thrombin, and without active thrombin chemistry, the fibrinogen/platelet cross link will become unstable, leading to disaggregation by dethrombosis.

7) Is this the same effect that works in Viagra for dilating an artery?

Both mechanisms depend on the availability of nitric oxide, but Viagra competitively inhibits an enzyme (phosphodiesterase Type 5; PDE-5), which is located specifically in the penile vasculature. PDE-5 destroys cyclic guanylate monophosphate (cGMP) but Viagra, by binding to PDE-5, preserves cGMP by taking its place. In both cases, cGMP is the product of nitric oxide interacting with the enzyme guanylate cyclase, resulting in smooth muscle cell relaxation and thus arterial dilation. The question of whether NO produced by UV light could override the effects of PDE-5 has never been addressed, but we conjecture this could be true although maybe impractical.

8) Are there new drugs being developed to go with UV facilitated dethrombosis?

The only drugs which have shown capability to disaggregate platelet aggregates are thrombin inhibitors but, as explained above, this was in the context of animal clot models in which the manner of making the clots (severely pinching the artery with forceps 3 times) was very crude. The thrombotic response to this injury was very slow (30 - 45 minutes) and non-uniform, and the resulting clots were not truly occlusive because they were full of seepage channels which permitted the entry of drugs. However, the clots extracted by Marder et al from humans contained no seepage channels. Such “subtleties,” if overlooked, likely account for a great deal of the mismatch between animal research and clinical relevance.

It is also important to mention that occluded arterial segments treated directly with UV do not rethrombose. This remarkable effect is in total contrast to every other form of clot removal, and is apparently due to the florid, highly localized production of NO by the UV laser. tPA, in contrast, exposes thrombin receptors on platelets, and this is often followed by re-occlusion owing to the activation of new platelets. There thus appears to be “no need” whatsoever to accompany dethrombosis with concurrent antiplatelet drugs, as is often done with tPA.

9) Are there any stroke victims that cannot be helped by the Opus process?

Severe hemorrhages per se cannot be treated by any method which increases blood flow to those same locations; however vasoconstriction resulting from hemorrhage certainly can be treated (viz. the Opus Univ. of Penn large animal study). Regarding those cases in which non-destructive, reversible arterial dilation can be beneficial, this method requires that smooth muscle cells be responsive to the UV irradiation. And in our experience, arteries which have been severely constricted by hemorrhage-induced vasospasm, or by mechanical trauma, or by occlusive thrombosis, all have been able to respond very satisfactorily, regardless of prior mechanical or photochemical damage to endothelium or even presumably to smooth muscle cells mediated by hemoglobin (a known scavenger of nitric oxide). At the minimum, we thus expect that any application of low power UV laser light to the arterial wall will lead to dilation, which could be critical if the occlusion consists of plaque fragments (i.e., properties of calcium).

10) Can this be used with the drugs that are used in treating stroke victims in the catheterization lab at the hospital?

This method will definitely enhance the efficacy of tPA, in particular, because dethrombosis creates a multitude of perfusion channels throughout the platelet matrix, rather than just a few large cracks induced presumably by ultrasonic augmentation. With such enhanced proximity to fibrin, the efficacy of tPA should be markedly enhanced, and we have indeed observed this ourselves in the case of reteplase. In view of this fundamental property of microchannel formation, we believe that tPA should be secondary to dethrombosis, not vice versa. Also, heparin is totally unnecessary as an adjunct, because platelet aggregation via GPIIb-IIIa cross-links is independent of its action.

11) What are the contra indications?

If a patient’s arteries are suspected of being extremely fragile and inelastic, along with loss of endothelial NO synthesis (observed as a concomitant of aging), the UV method may be compromised. However, this “fragility” is a standard contra indication for all current methods approved for marketing by the FDA.

12) How does this compare to using a laser to "blast" the clot into pieces?

UV laser-facilitated dethrombosis is the only published method known to OpusGen in which the laser beam is not required to be aimed directly at the arterial obstruction, regardless of its composition. Indeed, platelets are notorious scatterers of light, and when aggregated, form white clots which by definition reflect light of all colors. Because there is no intrinsic chromophore to absorb light energy, it is inherently pointless to try to explode such a clot by photoacoustic shock induced by high-intensity laser pulses. The power of UV laser-facilitated dethrombosis rests on the realization that the artery has within itself the substrate required for its rescue from occlusion, namely nitric oxide; NO need only be released from its stored forms and that is what the UV laser does. The power density required is orders of magnitude less than the lasers used previously. Of course, after dethrombosis there can be residue such as fibrin, but this certainly will not be sufficient to impede flow and can easily be removed with tPA, or even condensed by stent emplacement.

Practical considerations: Clots in humans are said to be bordered proximally and distally by agglutinated red blood cells over distances of the order of one diameter. UV light undergoes extreme attenuation in blood, so areas covered by red blood cell agglutinates must be avoided or else cleared before irradiation. “Cleared” in the case of flowing blood means that the blood must be diluted by a factor of ca. 1000 for UV light to effectively reach the inner arterial wall from the conical-tip optical fiber. Therefore, treatment cannot be begun if neither agglutinated or non-coagulated, flowing blood can be cleared (by saline infusion) from the arterial segment proximal to the clot. However, this requirement has readily been met by using standard accessories and standard methods practiced by interventional neuroradiology clinicians treating stoke intra-arterially.

13) How do you know that nothing goes downstream to cause more problems such as re-clotting?

In our animal research studies, we have found that dethrombosis inherently facilitates expansion of the part of the thrombus composed of aggregated platelets. In humans, the aggregated platelets appear in layers oriented transverse to the direction of flow (lines of Zahn), and we suspect that they will expand radially in human clots just as they have done in rat clots. The published photochemical clot model was used to test dethrombosis in rats represents a thick version of the same platelet layers found clinically in humans.

As explained previously, rethrombosis does not occur in an arterial segment formerly occluded by platelets and re-canalized by UV-facilitated dethrombosis. Clinically, as shown by Marder et al in 2006, atherothrombi (“white” clots) are indistinguishable from cardiogenic emboli (“red” clots); this report undermined 4 decades of publications regarding the origins of these clots. However, in the experience of neurointerventionalists, the densities of these clots may differ somewhat. Enlodged emboli are said to be compacted by the pressure of flowing blood, and although this results in mechanically harder clots presumably owing to increased platelet binding, it should present no difficulties to dethrombosis by Opus.